Purification and characterization of cytotoxic lymphocyte maturation factor and monoclonal antibodies thereto

ABSTRACT

The present invention is a novel cytokine protein called IL-12 or Cytotoxic Lymphocyte Maturation Factor (CLMF) which is produced and synthesized by human NC-37 B lymphoblastoid cells (American Type Culture Collection, Rockville, Md.). CLMF synergistically induces with low concentrations of IL-2 the cytolytic activity of Lymphokine Activated Killer (LAK) cells, and CLMF is capable of stimulating T-cell growth. Also claimed are the cloned gene for CLMF, its recombination in a suitable vector, the transformed cells containing said vector, the recombinant protein produced by the transformed cells and antibodies to CLMF.

This is a division, of application Ser. No. 08/205,011, filed Mar. 2,1994 (now abandoned); which is a divisional application of Ser. No.07/857,023, filed Mar. 24, 1992 (now abandoned); which is a CIP of Ser.No. 07/572,284, filed Aug. 27, 1990 (now abandoned); which is a CIP ofSer. No. 07/520,935, filed May 9, 1990 (now abandoned); which is a CIPof Ser. No. 07/455,708, filed Dec. 22, 1989 (now abandoned); and amendthe claims pursuant to the Preliminary Amendment submitted concurrentlyherewith.

FIELD OF THE INVENTION

The present invention relates to the field of cytokines, in particularto those cytokines which synergize with Interleukin-2 (IL-2) to activatecytotoxic lymphocytes.

BACKGROUND OF THE INVENTION

‘Cytokine’ is one term for a group of protein cell regulators, variouslycalled lymphokines, monokines, interleukins and interferons, which areproduced by a wide variety of cells in the body, play an important rolein many physiological responses, are involved in the pathophysiology ofa range of diseases, and have therapeutic potential. This heterogeneousgroup of proteins has the following characteristics in common. They arelow molecular weight (≦80 kDa) secreted proteins which are oftenglycosylated; are involved in immunity and inflammation where theyregulate the amplitude and duration of a response; and are usuallyproduced transiently and locally, acting in a paracrine or autocrine,rather than endocrine manner. Cytokines are extremely potent, generallyacting at picomolar concentrations; and interact with high affinity cellsurface receptors specific for each cytokine or cytokine group. Theircell surface binding ultimately leads to a change in the pattern ofcellular RNA and protein synthesis, and to altered cell behavior.Individual cytokines have multiple overlapping cell regulatory actions.

The response of a cell to a given cytokine is dependent upon the localconcentration of the cytokine, the cell type and other cell regulatorsto which it is concomitantly exposed. The overlapping regulatory actionsof these structurally unrelated proteins binding to different cellsurface receptors is at least partially accounted for by the inductionof common proteins which can have common response elements in their DNA.Cytokines interact in a network by: first, inducing each other; second,transmodulating cytokine cell surface receptors and third, bysynergistic, additive or antagonistic interactions on cell function.[Immunology Today 10 No. 9 p 299 (1989)].

The potential utility of cytokines in the treatment of neoplasia and asimmunoenhancing agents has recently been demonstrated in studies usinghuman recombinant interleukin-2 (rIL-2). Natural Interleukin-2 (IL-2) isa lymphokine which is produced and secreted by T-lymphocytes. Thisglycoprotein molecule is intimately involved in the induction ofvirtually all immune responses in which T-cells play a role. B cellresponses in vitro are also enhanced by the presence of IL-2, and IL-2has also been implicated as a differentiation inducing factor in thecontrol of B and T lymphocyte responses.

Administration of human rIL-2 has been shown in some cases to result inregression of established tumors in both experimental animals [J. Exp.Med 161:1169-1188, (1985)] and in man [N. Engl. J. Med 313: 1485-1492,(1985) and N. Engl. J. Med 316:889-897 (1987)]. The anti-tumor effectsof rIL-2 are thought to be mediated by host cytotoxic effectorlymphocytes which are activated by rIL-2 in vivo [J. Immunol.139:285-294 (1987)]. In addition, results from animal models suggestthat rIL-2 might also have value in the treatment of certain infectiousdiseases [J. Immunol. 135:4160-4163 (1985) and J. Virol. 61:2120-2127(1987)] and in ameliorating chemotherapy-induced immunosuppression[Immunol Lett. 10:307-314 (1985)].

However, the clinical use of rIL-2 has been complicated by the seriousside effects which it may cause [N. Engl. J. Med. 313:1485-1492 (1985)and N. Engl. J. Med. 316:889-897 (1987)]. One approach to improving theefficacy of cytokine therapy while reducing toxicity is to use two ormore cytokines in combination. For example, synergistic antitumoractivity has been shown to result when rIL-2 is administered totumor-bearing mice together with recombinant interferon alpha (rIFNalpha) [Cancer Res. 48:260-264 (1988) and Cancer Res. 48:5810-5817(1988)] or with recombinant tumor necrosis factor alpha (rTNFalpha)[Cancer Res. 47:3948-3953 (1987)]. Since the antitumor effects ofIL2 are thought to be mediated by host cytotoxic effector lymphocytes,it would be of interest to identify and isolate novel cytokines whichsynergize with rIL2 to activate cytotoxic lymphocytes in vitro. Thesenovel cytokines would also be useful as antitumor agents whenadministered in combination with rIL2 in vivo.

SUMMARY OF THE INVENTION

The present invention is a novel cytokine protein called CytotoxicLymphocyte Maturation Factor (CLMF) also called IL-12 which is producedand synthesized by cells capable of secreting CLMF such as mammaliancells particularly human NC-37 B lymphoblastoid cells (ATCC CCL 214American Type Culture Collection, Rockville, Md.). CLMF synergisticallyinduces with low concentrations of IL-2 (IL-2) the cytolytic activity ofLymphokine Activated Killer (LAK) cells, and CLMF is capable ofstimulating T-cell growth.

The present invention is directed toward the process of isolating CLMFin a substantially pure form.

The process comprises the following:

stimulating NC-37 B lymphoblastoid cells to produce and secretecytokines into a supernatant liquid;

collecting the supernatant liquid produced by the stimulated cells;

separating the supernatant liquid into protein fractions;

testing each protein fraction for the presence of CLMF;

retaining the protein fractions which are able to stimulate T-cellgrowth, said fractions containing an active protein which is responsiblefor the T-cell stimulating activity of the protein fractions;

isolating said active protein into a substantially pure form, saidprotein being Cytolytic Lymphocyte Maturation Factor (CLMF). CLMF is a75 kilodalton (kDa) heterodimer comprised of two polypeptide subunits, a40 kDa subunit and a 35 kDa subunit, which are bonded together via oneor more disulfide bonds.

The process of this invention is capable of purifying CLMF from anyliquid or fluid which contains CLMF together with other proteins. Alsoclaimed are the protein fractions capable of stimulating T-cell growth,the substantially purified active protein, CLMF, obtained from the abovedescribed process, the isolated cloned genes encoding the 40 kDa subunitand the 35 kDa subunit, vectors containing these genes and host cellstransformed with the vectors containing the genes.

In addition a method for stimulating LAK cells and T-cells comprised oftreating these cells with CLMF alone or with IL-2 is claimed. Alsoclaimed are isolated antibodies capable of binding to CLMF.

Antibodies to CLMF

Monoclonal antibodies prepared against a partially purified preparationof CLMF have been identified and characterized by 1: immunoprecipitationof ¹²⁵I-labelled CLMF, 2: immunodepletion of CLMF bioactivity, 3:western blotting of CLMF, 4: inhibition of ¹²⁵I-CLMF binding to itscellular receptor and 5: neutralization of CLMF bioactivity. Twentyhybridomas were identified which secreted anti-CLMF antibodies. Theantibodies immunoprecipitate ¹²⁵I-labelled CLMF and immunodeplete CLMFbioactivity as assessed in the T-cell proliferation and LAK cellinduction assays. Western blot analysis demonstrate that each antibodybinds to the 70 kDa heterodimer and to the 40 kDa subunit. These datademonstrated that the 20 antibodies were specific for CLMF and inparticular for the 40 kDa subunit of CLMF. A CLMF receptor binding assayhas been developed to evaluate the ability of individual antibodies toinhibit CLMF binding to its cellular receptor. The assay measures thebinding of ¹²⁵I-labelled CLMF to PHA activated PBL blast cells in thepresence and absence of each antibody. Of the 20 antibodies tested, 12antibodies inhibit greater than 60% of the ¹²⁵I-labelled CLMF binding tothe blast cells. Two inhibitory antibodies, 7B2 and 4A1, neutralize CLMFbioactivity while one non-inhibitory antibody, 8E3, does not neutralizeCLMF bioactivity. These data confirm that antibodies which block¹²⁵I-labelled CLMF binding to its cellular receptor will neutralize CLMFbioactivity as assessed by the T-cell proliferation and LAK cellinduction assays. The ability of the antibodies specific for the 40 kDasubunit of CLMF to neutralize CLMF bioactivity indicates thatdeterminants on the 40 kDa subunit are necessary for binding to the CLMFcellular receptor.

Utility for the Monoclonal Anti-Human CLMF Antibodies

The monoclonal anti-CLMF antibodies provide powerful analytical,diagnostic and therapeutic reagents for the immunoaffinity purificationof natural and recombinant human CLMF, the development of human CLMFimmunoassays, the identification of the active site of the 40 kDasubunit of CLMF and as possible therapeutic treatments which requireselective immunosuppression of cytotoxic T cells, such as intransplantation. Monoclonal antibodies which recognize differentepitopes on human CLMF can be used as reagents in a sensitive two-siteimmunoassay to measure levels of CLMF in biological fluids, cell culturesupernatants and human cell extracts.

The present invention is directed to monoclonal antibodies against CLMFwhich exhibit a number of utilities including but not limited to:

1. Utilizing the monoclonal antibodies as affinity reagents for thepurification of natural and recombinant human CLMF;

2. Utilizing the monoclonal antibodies as reagents to configureenzyme-immunoassays and radioimmunoassays to measure natural andrecombinant CLMF in biological fluids, cell culture supernatants, cellextracts and on plasma membranes of human cells and as reagents for adrug screening assay;

3. Utilizing the monoclonal antibodies as reagents to constructsensitive two-site immunoassays to measure CLMF in biological fluids,cell culture supernatants and human cell extracts;

4. Utilizing the monoclonal antibodies as reagents to identifydeterminants of the 40 kDa subunit which participate in binding to the35 kDa subunit and which participate in binding to the CLMF cellularreceptor;

5. Utilizing the intact IgG molecules, the Fab fragments or thehumanized IgG molecules of the inhibitory monoclonal antibodies astherapeutic drugs for the selective blockade of proliferation andactivation of cytotoxic T cells, such as in transplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of a supernatant solution obtained from cultured NC37lymphoblastoid cells applied to a Nu-Gel P-SP column showing the proteinfraction containing TGF activity being eluted with a salt gradient.

FIG. 2 is a plot of the material containing TGF activity obtained fromthe separation of FIG. 1 as it was being eluted with a salt gradientthrough a Blue-B-Agarose Column.

FIG. 3 shows the plot of the material containing TGF activity obtainedfrom the separation of FIG. 2 as it was being eluted with a NaClgradient through a Mono Q column.

FIG. 4 shows the SDS-polyacrylamide gel electrophoresis (SDS-PAGE)analysis of the fractions obtained from the step illustrated in FIG. 3.

FIG. 5 shows the elution profile through a Vydac Diphenyl column offraction 38 from the Mono Q Chromatography separation.

FIG. 6 shows SDS-PAGE analysis of protein purity of the proteinfractions 86-90 recovered from the process depicted in FIG. 5.

FIG. 7 shows the SDS PAGE analysis of fractions 87 and 88 undernon-reducing (without β-mercaptoethanol) and reducing (in the presenceof β-mercaptoethanol) conditions showing the 75,000 molecular weightCLMF separated into two subunits of 40 kDa and 35 kDa.

FIG. 8 shows the elution pattern of the proteins from the supernatantsolution from NC-37 cells applied to a Nu-Gel P-SP column eluted with asalt gradient in Example 2.

FIG. 9 is a Blue-B-Agarose column salt gradient elution profile of theactive fractions obtained from the Nu-Gel P-SP column elution of FIG. 8.

FIG. 10 is a Mono-Q column salt gradient elution profile of the activefractions obtained from the elution shown in FIG. 9.

FIG. 11 is the elution pattern through a Vydac Diphenyl column of activefractions 39 and 40 obtained from the Mono Q Chromatography shown inFIG. 10.

FIG. 12 is the SDS PAGE analysis under reducing conditions of the activefractions obtained from the elution of FIG. 11.

FIG. 13 is a schematic diagram showing the separation of the 40 kDasubunit from the 35 kDa subunit of the CLMF cytokine of the presentinvention.

FIG. 14 is a schematic diagram showing the determination of the aminoacid composition, N-terminal sequencing, proteolytic digestion andcomplete sequencing of the 40 kDa subunit of the CLMF cytokine of thepresent invention.

FIG. 15 is a tryptic peptide map of the digested 40 kDa subunit of theCLMF cytokine of the present invention.

FIG. 16 is a proteolytic peptide map of the digested 40 kDa subunit CLMFin which the proteolytic enzyme which was used was Staphylococcus aureusV8 protease.

FIG. 17 is a chart which summarizes the protein structural determinationof the 40 kDa subunit of CLMF.

FIG. 18 is the SDS PAGE analysis of Fraction 39 from the Mono Q FPLCelution shown in FIG. 4.

FIG. 19 is the elution pattern through a Vydac C-18 column of fraction39 of the Mono Q chromatography which was reduced in 5%β-mercaptoethanol.

FIG. 20 is the SDS-PAGE gel analysis under non-reducing conditions ofthe fractions which were fluorescamine positive from the Vydac C-18column elution shown in FIG. 19.

FIG. 21 is the elution pattern through a YMC ODS column of a trypticdigest of fractions 36 and 37 of the Mono Q Chromatography.

FIG. 22 shows a summary of the sequences obtained from CNBr fragments ofCLMF produced in Example 6.

FIG. 23 shows the reverse-phase HPLC of peptide fragments of CLMFproduced according to Example 6.

FIG. 24 is an SDS PAGE of pure CLMF and “free” unassociated 40 kDasubunit of CLMF purified by Affinity Chromatography according to Example7.

FIGS. 25A-D show the DNA and deduced amino acid sequences for the 40 kDasubunit of CLMF.

FIGS. 26A-C show the cDNA sequence and deduced amino acid sequence forthe human 35 kDa CLMF subunit.

FIG. 27 shows the inhibition of CLMF bioactivity by serum from CLMFimmunized and control rats.

FIG. 28 shows SDS PAGE analysis of immunoprecipitation of ¹²⁵I-CLMF bymonoclonal antibodies 4A1 (1), 4D1 (2), 8E3 (3), 9C8 (4) and control (5)and by immune rat serum (6 and 8) and normal rat serum (7 and 9).

FIG. 29 shows the immunodepletion of CLMF bioactivity (TGF activity) bymonoclonal anti-CLMF antibodies (a-CLMF).

FIG. 30 shows the immunodepletion of CLMF bioactivity (LAK inductionactivity) by monoclonal anti-CLMF antibodies (a-CLMF).

FIG. 31 shows Western blot analysis of the reactivity of monoclonal andrat polyclonal anti-CLMF antibodies with CLMF 75 kDa heterodimer.

FIG. 32 shows Western blot analysis of the reactivity of monoclonal andrat polyclonal anti-CLMF antibodies with CLMF 40 kDa subunit.

FIG. 33 shows the binding of ¹²⁵I-CLMF to PHA activated peripheral bloodlymphocyte (PBL) lymphoblasts.

FIG. 34 shows the inhibition of ¹²⁵I-CLMF binding to PHA-activated PBLblast cells by rat anti-CLMF serum. The data are expressed as amount (%bound) of ¹²⁵I-CLMF binding to the cells in the presence of theindicated concentrations of serum when compared to the total specificbinding in the absence of serum.

FIG. 35 shows the inhibition of ¹²⁵I-CLMF binding to PHA-activated PBLblast cells by monoclonal antibody supernatants. The data are expressedas % inhibition of ¹²⁵I-CLMF binding to the cells in the presence of a1:1 dilution of supernatant when compared to the total specific bindingin the absence of antibody supernatant.

FIG. 36 shows the inhibition of ¹²⁵I-CLMF binding to PHA-activated PBLblast cells by various concentrations of purified monoclonal antibodies.The data are expressed as the amount (% cpm bound) of ¹²⁵I-CLMF bindingto the cells in the presence of the indicated concentrations of antibodywhen compared to the total specific binding in the absence of antibody.

FIG. 37 shows Western blot analysis of the reactivity of a rabbitpolyclonal anti-CLMF antibody with 75 kDa CLMF (nonreduced) and with 35kDa CLMF subunit (reduced). The antibody was prepared against asynthetic peptide fragment of the 35 kDa CLMF subunit.

FIG. 38 shows the effect of IL-12 on IgE and IFN-γ production byIL-4-stimulated PBMC.

A. PBMC were cultured for 12 days in the presence of 10 ng/ml of IL-4and increasing concentrations of IL-12 (0.1 to 100 pM). Shown are themean ±1 SEM of three experiments; *significantly different from controlwithout IL-2 at P<0.01, Student's test.

B. Cells were cultured in the presence of IL-4 and IL-12 (60 pM), withor without anti-IL-12 mAbs (a mixture of antibodies 4A1 and 20C2, eachat 10 μg/ml).

FIG. 39 shows the IL-12 suppresses the accumulation of productive butnot germ-line Cε transcripts. Total RNA was extracted from PBMC culturedfor 10 days with 10 ng/ml of IL-4, in the absence (lanes 1, 3 and 5) orin the presence of 60 pM IL-12 (lanes 2, 4, and 6). Northern blot wasperformed as described. The membrane was hybridized with ³²P-labeledprobes specific for the germ-line transcript (lanes 1 and 2) or for theCε₁-Cε₂ region binding to both germ-line and mature Cε transcripts(lanes 3 and 4). Equal loading was assessed by methylene blue stainingof the ribosomal RNA (lanes 5 and 6).

DETAILED DESCRIPTION OF THE INVENTION

All publications mentioned herein, both supra and infra, are herebyincorporated herein by reference.

The CLMF (or IL-2) active proteins of the present invention include thehomogenous natural CLMF protein as well as CLMF proteins which contain abiologically active fragment of the amino acid sequence of natural CLMFand CLMF proteins which contain the amino acid sequence of natural CLMFtogether with other amino acids. The proteins of this invention have thebiological activity of CLMF as measured by standard assays such asT-cell growth factor assay as described in Example 9.

The protein of the present invention also include proteins which containanalogous amino acid sequences to CLMF or its CLMF active fragments.Such analogues are proteins in which one or more of the amino acids ofnatural CLMF or its fragments have been replaced or deleted withouteliminating CLMF activity. Such analogues may be produced by knownmethods of peptide chemistry or by known methods of recombinant DNAtechnology, such as planned mutagenesis. The CLMF biological activity ofall of the proteins of the present invention including the fragments andanalogues may be determined by using the standard T-cell growth factorassay such as described in Example 9.

In accordance with this present invention, natural CLMF is obtained inpure form. The amino acid sequence of this protein is depicted in FIGS.25 and 26. From the sequence of this protein obtained in accordance withthis invention, biologically active analogues and fragments of this CLMFprotein can be obtained. These biologically active proteins may beproduced biologically through standard recombinant technology or may bechemically synthesized using the sequence described above and an aminoacid synthesizer or manual synthesis using chemical conditions wellknown to form peptide bonds between selected amino acids. In thismanner, these analogues, fragments and proteins which contain the aminoacid sequence of CLMF together with other amino acids can be produced.All of these proteins may then be tested for CLMF activity.

Cytotoxic Lymphocyte Maturation Factor of the present invention wasisolated and purified as follows:

Production of Supernatant Liquid Containing CLMF

Human NC-37 B lymphoblastoid cells ATCC CCL 214 (American Type CultureCollection, Rockville, Md.) were used for production of CLMF. Thesecells were maintained by serial passage in RPMI 1640 medium supplementedwith 5% heat-inactivated (56° C., 30 min.) fetal bovine serum, 2 mML-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (allobtained from GIBCO Laboratories, Grand Island, N.Y.).

Higher producer sublines of NC-37 cells were derived by limitingdilution cloning in liquid microcultures. Each well of three Costar3596® microplates (Costar Co., Cambridge, Mass.) received 100 μl of acell suspension containing five NC-37 cells/ml. The medium used for thecloning was a 1:1 mixture of fresh passage medium and filtered,conditioned medium from stock cultures of the parent NC-37 cells. Oneweek and two weeks after culture initiation each of the microcultureswas fed with 50 μl of the 1:1 mix of fresh and conditioned medium.Between 3 and 4 weeks after culture initiation the contests of wellscontaining clones of NC-37 cells were harvested and passed into largercultures.

When the number of cells in a given subline exceeded 1.4×10⁶, onemillion cells were stimulated to produce CLMF in 1 ml culturescontaining 3 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma ChemicalCo., St. Louis, Mo.) and 100 ng/ml calcium ionophore A23187 (Sigma).Supernatants were harvested from the cultures after 2 days, dialyzedagainst about 50 volumes of Dulbecco's phosphate buffered saline (Gibco)using SPECTROPOR® #1 tubing (Fisher Scientific) overnight with onechange of buffer and then for 4 hours against 50 volumes of RPMI 1640medium with 50 μg/ml of gentamicin (both from Gibco) and tested for CLMFby means of the T cell growth factor assay (see below). Three sublines,NC-37.89, NC-37.98, and NC-37.102, were identified which routinelyproduced CLMF at titers ≧4 times the titers produced by the parent NC-37cell line. Since cells from these three sublines produced CLMF atsimilar titers (≧800 units/ml), culture supernatants derived from thethree sublines were pooled from use as starting material for thepurification of CLMF.

Bulk production of CLMF was carried out in roller bottle cultures on aroller apparatus set at about 38 rpms (Wheaton Cell Production RollerApparatus Model II, Wheaton Instruments, Millville, N.J.). Cellsuspensions were prepared containing 1-1.5×10⁶ NC-37.89, NC-37.98 orNC-37.102 cells/ml of RPMI 1640 supplemented with 1% Nutridoma-SP(Boehringer Mannheim Biochemicals, Indianapolis, Ind.), 2 mML-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 10 ng/mlPMA and 20-25 ng/ml calcium ionophore A23187. Two hundred fifty to threehundred fifty ml aliquots of the cell suspensions were added to Falcon3027 tissue culture roller bottles (Becton Dickinson, Lincoln Park,N.J.) which has been gassed with a mixture of 5% CO₂, 95% air. Theroller bottles were then capped tightly and incubated at 37° C. withcontinuous rolling for three days. At the end of this time, the culturesupernatants were harvested. EDTA and phenylmethylsulfonyl fluoride(both from Boehringer Mannheim) were added to the culture supernatantsat final concentrations of 1 mM and 0.1 mM, respectively, to retardproteolytic degradation. The supernatants were stored at 4° C. untilconcentration.

Lympokine Activated Killer (LAK) Cell Induction (LCI) Assay

Culture supernatants and chromatographic fractions were tested for theirability to synergize with rIL-2 to induce the generation of cytolyticLAK cells as follows. Human peripheral blood mononuclear cells (PBMC)were isolated by the following method. Blood from normal volunteerdonors was drawn into syringes containing sufficient sterilepreservative-free heparin (Sigma) to give a final concentration ofapproximately 5 units/ml. The blood was diluted 1:1 with Hanks' balancedsalt solution (HBSS) without calcium or magnesium (GIBCO). The dilutedblood was then layered over 15 ml aliquots of Ficoll/sodium diatrizoatesolution (Lymphocyte Separation Medium, Organon Teknika Corp., Durham,N.C.) in 50 ml Falcon 2098 centrifuge tubes. The tubes were centrifugedfor 30 minutes at room temperature at 500×g. Following centrifugation,the cells floating on the Ficoll/sodium diatrizoate layer were collectedand diluted by mixing with ≧2 volumes of HBSS without calcium ormagnesium. The resulting cell suspension was then layered over 15 mlaliquots of 20% sucrose (Fisher) in RPMI 1640 medium with 1% human ABserum (Irvine Scientific, Santa Ana, Calif.) in Falcon 2098 centrifugetubes. The tubes were centrifuged for 10 minutes at room temperature at500×g, and the supernatant fluids were discarded. The cell pellets wereresuspended in 5 ml of HBSS without calcium or magnesium, repelleted bycentrifugation, and finally resuspended in the appropriate culturemedium. Accessory cells were removed from the PBMC by treatment with 5mM L-glutamic acid dimethyl ester (Sigma) using the same conditionsdescribed by Thiele et al. J. Immunol. 131:2282-2290, (1983) foraccessory cell depletion by L-leucine methyl ester except that theglutamic ester was substituted for the leucine ester.

The accessory cell-depleted PBMC were further fractionated bycentrifugation on a discontinuous Percoll density gradient (Pharmacia,Piscataway, N.J.) as described by Wong et al., Cell Immunol. 111:39-54,(1988). Mononuclear cells recovered from the 38, 41, 45, and 58% Percolllayers were pooled and used as a source of LAK cell precursors in theassay. The cells recovered from the Percoll gradient were washed andsuspended in tissue culture medium (TCM) composed of a 1:1 mixture ofRPMI 1640 and Dulbecco's modified Eagle's medium, supplemented with 0.1mM nonessential amino acids, 60 μg/ml arginine HCl, 10 mM HEPES buffer,2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin (allavailable from GIBCO), 5×10⁻⁵ M 2-mercaptoethanol (Fisher Scientific,Fair Lawn, N.J.), 1 mg/ml dextrose (Fisher), and 5% human AB serum(Irvine Scientific, Santa Ana, Calif.). These cells were incubated in24-well tissue culture plates (Costar, Cambridge, Mass.) in 1 mlcultures (7.5×10⁵ cells/culture) to which 10⁻⁴ M hydrocortisone sodiumsuccinate (Sigma) was added to minimize endogenous cytokine production.Some cultures also received human rIL-2 (supplied by Hoffmann-La Roche)at a final concentration of 5 units/ml and/or supernatants to be assayedfor CLMF activity. All cultures were incubated for 3-4 days at 37° C. ina humidified atmosphere of 5% CO₂, 95% air.

At the end of this incubation, the contents of each culture wereharvested, and the cells were pelleted by centrifugation and resuspendedin 0.5 ml of fresh TCM. One tenth ml aliquots of these cell suspensionswere mixed with 0.1 ml aliquots of ⁵¹Cr-labelled K562 or Raji cells(both cell lines obtained from the ATCC) and tested for their lyticactivity in 5 hour ⁵¹Cr release assays. The method for labelling targetcells with ⁵¹Cr and performing the cytolytic assays have been describedby Gately et al., [JNCI 69:1245-1254 (1982)]. The percent specific ⁵¹Crrelease was calculated as [(e−c)/100−c)]×100, where e is the percentageof ⁵¹Cr released from target cells incubated with lymphocytes and c isthe percentage of ⁵¹Cr released spontaneously from target cellsincubated alone. The total releasable ⁵¹Cr was determined by lysis ofthe target cells with 2% sodium dodecyl sulfate; see Gately et al., JNCI69:1245-1254 (1982). All lymphocyte populations were assayed inquadruplicate for lytic activity.

LAK Cell Induction Microassay

The microassay for measuring synergy between rIL-2 and CLMF-containingsolutions in the induction of human LAK cells was similar to the LAKcell induction assay described above but with the followingmodifications. Human peripheral blood mononuclear cells which had beendepleted of accessory cells and fractionated by Percoll gradientcentrifugation as described above were added to the wells of Costar 3596microplates (5×10⁴ cells/well). Some of the wells also received rIL-2 (5units/ml final concentration) and/or purified CLMF or immunodepletedCLMF-containing solutions. All cultures contained 10⁻⁴ M hydrocortisonesodium succinate (Sigma) and were brought to a total volume of 0.1 ml byaddition of TCM with 5% human AB serum. The cultures were incubated for3 days at 37° C., after which 0.1 ml of ⁵¹Cr-labelled K562 cells (5×10⁴cells/ml in TCM with 5% human AB serum) were added to each well. Thecultures were then incubated overnight at 37° C. Following this, thecultures were centrifuged for 5 minutes at 500×g, and the supernatantsolutions were harvested by use of a Skatron supernatant collectionsystem (Skatron, Sterling, Va.). The amount of ⁵¹Cr released into eachsupernatant solution was measured with a gamma counter (Packard,Downer's Grove, Ill. ), and the % specific ⁵¹Cr release was calculatedas described above. All samples were assayed in quadruplicate.

Cytolytic T Lymphocyte (CTL) Generation Assay

Methods used for generating and measuring the lytic activity of humanCTL have been described in detail in Gately et al., J. Immunol. 136:1274-1282, 1986, and Wong et al., Cell. Immunol. 111:39-54, 1988. Humanperipheral blood mononuclear cells were isolated from the blood ofnormal volunteer donors, depleted of accessory cells by treatment withL-glutamic acid dimethyl ester, and fractioned by Percoll gradientcentrifugation as described above. High density lymphocytes recoveredfrom the interface between the 45% and 58% Percoll layers were used asresponder lymphocytes in mixed lymphocyte-tumor cultures (MLTC). CTLwere generated in MLTC in 24-well tissue culture plates (Costar #3424)by incubation of Percoll gradient-derived high density lymphocytes(7.5×10⁵/culture) together with 1×10⁵ uv-irradiated HT144 melanoma cells(ATCC, Rockville, Md.) or with 5×10⁴ gamma-irradiated HT144 melanomacells in TCM with 5% human AB serum (1.2 ml/culture). Foruv-irradiation, HT144 cells were suspended at a density of 1-1.5×10⁶cells/ml in Hanks' balanced salt solution without phenol red (GIBCO)containing 1% human AB serum. One ml aliquots of the cell suspensionwere added to 35×10 mm plastic tissue culture dishes (Falcon #3001), andthe cells were then irradiated (960 μW/cm² for 5 min) by use of a 254 nmuv light (model UVG-54 MINERALIGHT® lamp, Ultra-violet Products, Inc.,San Gabriel, Calif.). For gamma irradiation, HT144 cells were suspendedat a density of 1-5×10⁶ cells/ml in TCM with 5% human AB serum andirradiated (10,000 rad) by use of a cesium source irradiator (model 143,J. L. Shepherd and Associates, San Fernando, Calif.). Uv- orgamma-irradiated HT144 were centrifuged and resuspended in TCM with 5%human AB serum at the desired cell density for addition to the MLTC. Inaddition to lymphocytes and melanoma cells, some MLTC received humanrIL-2 and/or purified human CLMF at the concentrations indicated in thetable. Hydrocortisone sodium succinate (Sigma) was added to the MLTC ata final concentration of 10⁻⁴ M (cultures containing uv-irradiatedmelanoma cells) or 10⁻⁵ M (cultures containing gamma-irradiated melanomacells) to supress endogenous cytokine production [S. Gillis et al., J.Immunol. 123: 1624-1631, 1979] and to reduce the generation ofnonspecific LAK cells in the cultures [L. M. Muul and M. K. Gately, J.Immunol. 132: 1202-1207, 1984]. The cultures were incubated at 37° C. ina humidified atmosphere of 5% CO₂ in air for 6 days. At the end of thistime, lymphocytes from replicate cultures were pooled, centrifuged,resuspended in 1.2 ml TCM containing 5% human AB serum, and tested fortheir ability to lyse HT144 melanoma cells, and, as a specificitycontrol, K562 erythroleukemia cells in overnight ⁵¹Cr release assays.

Melanoma cells and K562 cells were labeled with ⁵¹Cr sodium chromate asdescribed by Gately et al. (JNCI 69: 1245-1254, 1982). Likewise,measurement of lymphocyte-mediated lysis of ⁵¹Cr-labeled melanoma cellswas performed in a manner identical so that described by Gately et al.(ibid.) for quantitating lysis of glioma target cells. For assaying thelysis of ⁵¹Cr-labeled K562 cells, 0.1 ml aliquots of lymphocytesuspensions were mixed with 25 μl aliquots or ⁵¹Cr-labeled K562 (2×10⁵cells/ml in TCM with 5% human AB serum) in the wells of Costar 3696“half-area” microtest plates. After overnight incubation at 37° C., theplates were centrifuged for 5 min at 1400×g, and 50 μl of culture mediumwas aspirated from each well. The amount of ⁵¹Cr in each sample wasmeasured with a gamma counter (Packard), and the % specific ⁵¹Cr releasewas calculated as described above. All assays were performed inquadruplicate, and values in the table represent the means ±1 S.E.M. ofreplicate samples.

T Cell Growth Factor (TGF) Assay

The ability of culture supernatants and chromatographic fractions tostimulate the proliferation of PHA-activated human T lymphoblasts wasmeasured as follows. Human PBMC were isolated by centrifugation overdiscontinuous Ficoll and sucrose gradients as described above for theLCI assay. The PBMC (5×10⁵ cells/ml) were cultured at 37° C. in TCMcontaining 0.1% phytohemagglutinin-P (PHA-P) (Difco Laboratories,Detroit, Mich.). After 3 days, the cultures were split 1:1 with freshTCM, and human rIL-2 was added to each culture to give a finalconcentration of 50 units/ml. The cultures were then incubated for anadditional 1 to 2 days, at which time the cells were harvested, washed,and resuspended in TCM at 4×10⁵ cells/ml. To this cell suspension wasadded heat-inactivated goat anti-human rIL-2 antiserum (final dilution:1/200) to block any potential IL-2-induced cell proliferation in theassay. This antiserum, which was provided by R. Chizzonite, MolecularGenetics Department, Hoffmann-La Roche, was shown to cause 50%neutralization of 2 units/ml rIL-2 at a serum dilution of 1/20,000. Anequally functional anti-human rIL2 antibody can be obtained from theGenzyme Co., Boston, Mass.

Fifty μl aliquots of the cell suspension containing anti-IL-2 antiserumwere mixed with 50 μl aliquots of serial dilutions of culturesupernatants or chromatographic fractions in the wells of Costar 35%microplates. The cultures were incubated for 1 day at 37° C. in ahumidified atmosphere of 5% CO₂ in air, and 50 μl of ³H-thymidine (NewEngland Nuclear, Boston, Mass.), 10 μCi/ml in TCM, were then added toeach well. The cultures were further incubated overnight. Subsequently,the culture contents were harvested onto glass fiber filters by means ofa cell harvester (Cambridge Technology Inc., Cambridge, Mass.), and³H-thymidine incorporation into cellular DNA was measured by liquidscintillation counting. All samples were assayed in triplicate.

In purifying CLMF it was necessary to define units of activity in orderto construct chromatographic elution profiles and to calculate thepercent recovery of activity and the specific activity of the purifiedmaterial. To do this, a partially purified preparation of humancytokines produced by coculturing PHA-activated human PBMC with NC-37cells was used as a standard. Several dilutions of this preparation,which was assigned an arbitrary titer of 2000 units/ml, were included ineach. TGF or LAK induction assay The results obtained for the standardpreparation were used to construct a dose-response curve from whichcould be interpolated units/ml of activity in each unknown sample at thedilution tested. Multiplication of this value by the dilution factoryielded the activity of the original sample expressed in units/ml.

For antibody neutralization studies, the TGF assay was modified asfollows: Twenty-five μl aliquots of CLMF-containing medium were mixedwith 50 μl aliquots of serial dilutions of antiserum or antibodysolutions in the wells of COSTAR 3596® microplates. The mixtures wereincubated for 30 min; at 37° C.; and 25 μl aliquots of a suspension ofPHA-activated lymphoblasts (8×10⁵/ml in TCM plus 1:100 anti-rIL-2) werethen added to each well. The cultures were further incubated, pulsedwith ³H-thymidine, harvested, and analyzed for ³H-thymidineincorporation as described above.

Natural Killer (NK) Cell Activation Assay

Purified CLMF was tested for its ability to activate NK cells when addedalone or in combination with rIL-2 as follows: Human PBMC were isolatedby centrifugation over discontinuous Ficoll and sucrose gradients asdescribed above and were suspended in RPMI 1640 medium supplemented with10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100μg/ml streptomycin, and 2 mM L-glutamine. The PBMC were incubatedovernight at 37° C. in 1 ml cultures (5×10⁶ cells/culture) together withrIL-2 and/or purified CLMF at various concentrations. After 18-20 hours,the contents of the cultures were harvested and centrifuged, and thecells were resuspended in the same medium used for the overnightcultures. The cytolytic activity of the cultured PBMC was then assessedin ⁵¹ Cr release assays as described above.

Purification and Characterization of Cytotoxic Lymphocyte MaturationFactor (CLMF)

Concentration of Cell Supernatant Solutions

Crude human CLMF supernatant solutions prepared from several batches ofinduced NC-37 cells were pooled and concentrated 30-fold using thePellicon Cassette System (30,000 NMWL PTTK00005 Millipore Corp. Bedford,Mass.), concentrating to the desired volume, a buffer exchange wasperformed with 10 mM MES, pH adjusted to 6.0 with 10N NaOH. Theconcentrate was centrifuged at 10;000×g for 10 min at 4° C. precipitatediscarded.

Ion-Exchange Chromatogrphy on NuGel P-SP Column

The concentrated supernatant solution was applied at a flow rate of 120ml/hr to a NuGel P-SP (Separation Industries, Metuchen, N.J.) column(5×5 cm), equilibrated in 10 mM MES, pH 6.0. The column was washed untilbaseline absorbance monitoring at 280 nm was obtained. Absorbed proteinswere then eluted with a 500 ml salt gradient from 0 to 0.5 M NaCl/10 mMMES, pH 6.0 at a flow rate of 2 ml/min. Aliquots of fractions wereassayed for TGF activity. Fractions containing TGF activity were pooledand dialyzed (Spectra/Por 7, Fisher Scientific) against 50 vol 20 mMTris/HCl, pH 7.5.

Dye-Affinity Chromatography on Blue B-Agarose Column

The dialyzed sample was centrifuged at 10,000×g for 10 min at 4° C. andthe precipitate discarded. The supernatant solution was applied at aflow rate of 20 ml/hr to a Blue H-Agarose (Amicon, Danvers, Mass.)column (2.5×10 cm) equilibrated in 20 mM Tris/HCl, pH 7.5. The columnwas washed with this same buffer until baseline absorbance monitoring at280 nm was obtained. Absorbed proteins were then eluted with a 500 mlsalt gradient from 0 to 0.5M NaCl/20 mM Tris/HCl, pH 7.5 at a flow rateof 15 ml/hr. Aliquots of fractions were assayed for TGF activity.Fractions containing TGF activity were pooled and dialyzed (Spectra/Por7, Fisher Scientific) against 100 vol 20 mM: Tris/HCl, pH 7.5.

Ion-Exchange Chromatography on Mono O Chromatography

The dialyzed sample was filtered through a 0.45 μm cellulose acetatefilter (Nalgene .Co.; Rochester, N.Y.) and the filtrate applied at aflow rate of 60 ml/hr to a Mono Q HR 5/5 (Pharmacia LKB Biotechnology;Inc., Piscataway, N.J.) column (5×50 mm) equilibrated in 20 mM Tris/HCl,pH 7.5. The column was washed with this same buffer until baselineabsorbance monitoring at 280 nm was obtained. Absorbed proteins werethen eluted with a 1 hr linear salt gradient from 0 to 0.25 M NaCl/20 mMTris/HCl, pH 7.5 at a flow rate of 60 ml/hr. Aliquots of fractions wereassayed for TGF activity and protein purity was assessed withoutreduction by SDS-PAGE [Laemmli, U.K. (1970) Nature (London) 227:680-685]using 12% slab gels. Gels were silver stained [Morrissey, Anal. Biochem.117:307-310] to visualize protein. Fractions which did not contain asingle band were further purified by reversed-phase HPLC.

Reversed-phase HPLC

The chromatographic system has been described previously by Stern, A. S.and Lewis, R. V. (1985) in Research Methods is Neurochemistry, Eds.Marks, N. and Rodnight, R. (Plenum, N.Y.) Vol. 6, 153-193. An automatedfluorescence detection system using fluorescamine (Polysciences, Inc.,Warrington, Pa.) monitored the protein in the column effluents [Stein,S. and Moschera, J. (1981) Methods Enzymol. 78:436-447]. Reversed-phaseHPLC was carried out using Vydac C18 or Biphenyl columns (4.6×20 mm, TheSep/a/ra/tions Group, Hesperia, Calif.). Proteins were eluted with anacetonitrile gradient in 0.1% TFA.

Protein Analysis

Amino acid analysis was performed on an instrument which usedpost-column reaction with fluorescamine for detection [Pan, Y.- C. E.,and Stein, S. (1986) in Methods of Protein Microcharacterization(Shively, J. E., Ed.), pp. 105-119, Humana Press,. Clifton, N.J.]

Sequence analysis was performed using an Applied Biosystems Inc. Model470A gas phase sequencer (Foster City, Calif.) [Hewick, R. M.,Henkapillar, M. W., Hood, L. E., and Dreyer, W. J. (1981) J. Biol. Chem256:7990-7997]. Phenylthiohydantoin (PTH) amino acid derivatives wereidentified “on-line” with an ABI Model 120A PTH analyzer.

Cloning of the CLMF Gene

As used herein, the term “CLMF polynucleotide containing a sequencecorresponding to a cDNA” means that the CLMF polynucleotide contains asequence which is homologous to or complementary to a sequence in thedesignated DNA; the degree of homology or complementary to the cDNA willbe approximately 50% or greater, will preferably be at least about 70%,and even more preferably will be at least about 90%. The correspondencebetween the CLMF sequences and the cDNA can be determined by techniquesknown in the art, including, for example, a direct comparison of thesequenced material with the cDNAs described, or hybridization anddigestion with single strand nucleases, followed by size determinationof the digested fragments.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.Maniatis, Fitsch & Sambrook, Molecular Cloning; A Laboratory Manual(1982); DNA Cloning, Volumes I AND II (D. N Glover ed. 1985)Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization (B. D. Homes & S. J. Higgins eds. 1984); Transcription andTranslation (B. D. Harnes & S. J. Higgins eds. 1984); Animal CellCulture (R. I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRLPress, 1986); B. Perbal, A. Practical Guide to Molecular Cloning (1984);the series, Methods in Enzymology (Academic Press, Inc.); Gene TransferVectors, for Mammalian Cells (J. H. in Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol.155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker,eds. (1987), Immunochemical Methods in cell and Molecular Biology(Academic Press, London), Scopes, (1987), Protein Purification:Principles and Practice, Second Edition (Springer-Verlag, N.Y.), andHandbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell eds 1986).

The DNA sequences and DNA molecules of the present invention may beexpressed using a wide variety of host/vector combinations. For example,useful vectors may consist of segments of chromosomal, non-chromosomaland synthetic DNA sequences, such as various known derivatives of SV-40and known bacterial plasmids, e.g., plasmids from E. coli includingpCR1, pBR322, pMB9 and RP4, phage DNAs, e.g., the numerous derivativesof phageg, and other DNA phages, e.g., M13 and other Filamentoussingle-stranded DNA phages, vectors useful in yeasts such as the 2mplasmid, vectors useful in eukaryotic cells, such as vectors useful inanimal cells, such as those containing SV-40 adenovirus and retrovirusderived DNA sequences and vectors derived from combinations of plasmidsand phage DNA's, such as plasmids which have been modified to employphage DNA or other derivatives thereof.

Such expression vectors are also characterized by at least oneexpression control sequence that may be operatively linked to the CLMFDNA sequence inserted in the vector in order to control and to regulatethe expression of that cloned DNA sequence. Examples of usefulexpression control sequences are the lac system, the trp system; the tacsystem, the trc system, major operator and promoter regions of phage λ,the control region of fd coat protein, the glycolytic promoters ofyeast, e.g., the promoter for 3-phosphoglycerate kinase, the promotersof yeast acid phosphatase, e.g., Pho 5, the promoters of the yeasta-mating factors, and promoters derived from polymoma, adenovirus,retrovirus, and simian virus, e.g., the early and late promoters orSV40, and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells and their viruses or combinationsthereof.

Among such useful expression vectors are vectors that enable theexpression of the cloned CLMF-related DNA sequences in eukaryotic hosts,such as animal and human cells [e.g., P. J. Southern and P. Berg; J.Mol. Appl. Genet.; 1, pp. 327-41 (1982); S. Subramani et. al., Mol. inCell. Biol., 1, pp. 854-64 (1981); R. J. Kaufmann and P. A. Sharp, Mol.Cell: Biol., 159, pp. 601-64 (1982) S. I. Scahill et al., “Expressionand Characterization of The Product Of A Human Immune Interferon DNAGene in Chinese Hamster Ovary Cells”, Proc. Natl. Acad. Sci. U.S.A., 80,pp. 4654-59 (1983); G. Urlaub and L. A. Chasin, Proc. Natl. Acad. Sci.USA, 77, pp. 4216-20 (1989)].

Furthermore, within each specific expression vector, various sites maybe selected for insertion of the CLMF-related DNA sequences of thisinvention. These sites are usually designated by the restrictionendonuclease which cut them. They are well recognized by those of skillin the art. It is, of course to be understood that an expression vectoruseful in this invention need not have a restriction endonuclease sitefor insertion of the chosen DNA fragment. Instead, the vector could bejoined to the fragment by alternative means. The expression vector, andin particular the site chosen therein for insertion of a selected DNAfragment and its operative linking therein to an expression controlsequence, is determined by a variety of factors, e.g., number of sitessusceptible to a particular restriction enzyme, susceptibility of theprotein to proteolytic degradation by host cell enzymes, contaminationof the protein to be expressed by host cell proteins difficult to removeduring purification; expression characteristics, such as the location ofstart and stop codons relative to the vector sequences, and otherfactors recognized by those of skill in the art. The choice of a vectorand an insertion site for a DNA sequence is determined by a balance ofthese factors, not all selections being equally effective for a givencase.

Not all host/expression vector combinations function with equalefficiency in expressing the DNA sequences of this invention or inproducing the CLMF polypeptides of this invention. However, a particularselection of a host/expression vector combination may be made by thoseof skill in the art after due consideration of the principles set forthherein without departing from the scope of this invention. For example,the selection should be based on a balancing of a number of factors.These include, for example, compatibility of the host and vector,toxicity of the proteins encoded by the DNA sequence to the host, easeof recovery of the desired protein, expression characteristics of theDNA sequences and the expression control sequences operatively linked tothem, biosafety, costs and the folding, form or any other necessarypost-expression modifications of the desired protein.

The CLMF produced by fermentation of the prokaryotic and eukaryotichosts transformed with the DNA sequences of this invention can then beemployed in the LAK cell and T cell activator and antitumor compositionsand methods of the present invention.

The CLMF of the present invention can also be analyzed to determinetheir active sites for producing fragments or peptides, includingsynthetic peptides, having the activity of CLMF. Among the knowntechniques for determining such active sites are x-ray crystallography,nuclear magnetic resonance, circular dichroism, UV spectroscopy and sitespecific mutagenesis. Accordingly, these fragments may be employed inmethods for stimulating T-cells or LAK cells.

Administration of the polypeptides, or perhaps peptides derived orsynthesized from them or using their amino acid sequences, or theirsalts or pharmaceutically acceptable derivatives thereof, may be via anyof the conventionally accepted modes of administration of agents whichexhibit antitumor activity. These include parenteral, subcutaneous,intravenous, or intralesional administration.

The preferred form of administration depends on the intended mode ofadministration on and therapeutic application. The compositions alsowill preferably include conventional pharmaceutically acceptablecarriers and may include other medicinal agents, carriers, adjuvants,excipients, etc., e.g., human serum albumin or plasma preparations.Preferably, the compositions of the invention are in the form of a unitdose and will usually be administered one or more times a day.

Methods for Assay of Monoclonal Antibodies

Purification of CLMF and Labelling on CLMF with ¹²⁵I.

CLMF was partially purified from cell supernatants harvested from humanperipheral blood lymphocytes (PBLs) or NC-37 cells as describedpreviously. Partially purified CLMF was labelled with ¹²⁵I by amodification of the Iodogen method (Pierce Chemical Co.). Iodogen(Pierce Chemical Co.) was dissolved in chloroform at a concentration of0.5 mg/ml and 0.1 ml aliquots were added to 12×75 borosilicate glasstubes. The chloroform was evaporated under a stream of nitrogen and theIodogen was dried in the center of the bottom of the glass tube. Thecoated tubes were stored in a dessicator at room temperature (RT) undervacuum. For radiolabeling 0.5-1.0 mCi ¹²⁵I-Na (Amersham) was added to anIodogen coated tube containing 0.50 ml of Tris-Iodination Buffer (25 mMTris-HCl pH 7.5, 0.4 M NaCl, 1 mM EDTA) and incubated for 4 minutes atRT. The activated ¹²⁵I solution was transferred to a 1.5 ml tubecontaining 0.05-0.1 ml CLMF (approximately 5 μg in 0.125 M NaCl, 20 mMTris-HCl pH 7.5) and the reaction was further incubated for 8 minutes atRT. At the end of the incubation, 0.05 ml of Iodogen Stop Buffer (10mg/ml tyrosine, 10% glycerol in Dulbecco's phosphate buffered saline(PBS) pH 7.4) was added and reacted for 30 seconds. The mixture was thendiluted with 1.0 ml Tris-Iodination Buffer and applied to a BioRadBioGel P10DG BioRad Laboratories) desalting column for chromatography.The column was eluted with Tris-Iodination Buffer and fractions (1 ml)containing the peak amounts of labelled protein were combined anddiluted to 1×10⁸ cpm/ml with 0.25% gelatin in Tri iodination buffer. TheTCA precipitable radioactivity (10% trichloroacetic final concentration)was typically in excess of 95% of the radioactivity. The radiospecificactivity ranged from 6000 cpm/fmol to 10,000 cpm/fmol.

Immunodepletion of CLMF

Hybridoma culture supernatants or purified monoclonal antibodies weretested for their ability to immunodeplete CLMF as follows: Goat anti-ratIgG-agarose beads (Sigma Chemical Co., St. Louis; Mo.) were washed threetimes with 10 ml of PBS (Gibco) supplemented with 1% bovine serumalbumin (BSA) (Sigma) (PBS/BSA solution). After washing, the beads wereresuspended in PBS/BSA at a final concentration of 50% vol/vol. Aliquots(0.2 ml) of the bead suspension were added to 1.5 ml Eppendorf tubes,together with the indicated amounts of monoclonal antibodies orhybridoma supernatant solutions. The volume of each mixture was broughtto 1.4 ml by the addition of hybridoma maintenance medium [Iscove'smodified Dulbecco's medium (IMDM) with 0.1% fetal bovine serum (FBS),10% Nutridoma-SP (Boehringer-Mannheim), and 2 mM L-glutamine], and themixtures were then incubated for 2 hours at room temperature on ahematology/chemistry mixer. Following this incubation, the tubes werecentrifuged in a Beckman microfuge 12 (1.5 minutes at setting 5), andthe supernatants were discarded. The beads were again washed three timeswith PBS/BSA and then resuspended in 1 ml of tissue culture medium (TCM)containing 5% human AB serum and the indicated concentration of purifiedhuman CLMF. The tubes were subsequently incubated overnight at 4° C. onthe mixer. Following this, the beads were removed by centrifugation inthe microfuge, and the resulting immunodepleted supernatant solutionswere assayed for residual CLMF activity in the TGF assay or in themicroassay for LAK cell induction.

Immunoprecipitation Assay

For the immunoprecipitation reaction, 0.05 to 0.5 ml of hybridomasupernatant, diluted antisera or purified IgG was added to a 1.5 mlmicrofuge tube containing 0.1 ml of a 50% suspension of goat-anti-ratIgG coupled to agarose (Sigma Chemical Co.). The assay volume wasbrought up to 0.5 ml with RIPA Buffer (50 mM NaPO₄ pH 7.5, 150 mM NaCl,1% Triton-X 100, 1% Deoxycholic acid, 0.1% SDS, 1% BSA, and 5 mM EDTA)and the mixture was incubated on a rotating mixer for 2 hours at RT. Thebeads were pelleted by centrifugation for 1 minute at 12,000×g and thenresuspended in 1 ml RIPA Buffer containing ¹²⁵I CLMF (1×10⁵ cpm). Themixture was then incubated on a rotating mixer for 16 hours at 4° C.Following this incubation, the beads were pelleted by centrifugation andwashed 2× in RIPA without BSA. The beads were then washed 1× with 0.125M Tris-HCl pH 6.8 and 10% glycerol. The ¹²⁵I-CLMF bound to the solidphase antibodies was released by adding 10 μl of 2× Laemmli SampleBuffer with and without 5% β-mercaptoethanol and heating for 3 minutesat 95° C. The immunoprecipitated 125I-CLMF was analyzed by SDS-PAGE on a10% or 12% polyacrylamide gel and visualized by autoradiography.

LCLMF Receptor Binding Assay

The ability of hybridoma supernatant solutions, purified IgG or antiserato inhibit the binding of ¹²⁵I-CLMF to PHA-activated human Tlymphoblasts was measured as follows: 0.1 ml aliquots of serialdilutions of culture supernatants, purified IgG or antisera were mixedwith 0.025 ml aliquots of Binding Buffer (RPMI-1640, 5% FBS, 25 mM HEPES-pH 7.4) containing ¹²⁵I-CLMF (1×10⁵ cpm). The mixture was incubated onan orbital shaker for 1 hour at RT, then 0.025 ml of activated blasts(5×10⁷ cells/ml) was added to each tube. The mixture was furtherincubated for 1 hour at RT. Non-specific binding was determined byinclusion of 10 nM unlabelled CLMF in the assay. Incubations werecarried out in duplicate or triplicate. Cell bound radioactivity wasseparated from free ¹²⁵I-CLMF by centrifugation of the assay contentsthrough 0.1 ml of an oil mixture (1:2 mixture of Thomas in SiliconeFluid 6428-R15: A. H. Thomas, and Silicone Oil AR 200:Gallard-Schlessinger) at 4° C. for 90 seconds at 10,000×g. The tipcontaining the cell pellet was excised and cell bound radioactivity wasdetermined in a gamma counter.

SDS Polyacrylamide Gel Electrophoresis (SDS/PAGE) and Western Blotting

Immunoprecipitated ¹²⁵I-labelled proteins and partially purified CLMFwere treated with Laemmli sample buffer (2% SDS, 125 mM Tris-HCl, pH6.8, 10% glycerol, 0.025% bromphenol blue) with and without 5%β-mercaptoethanol, heated at 95° C. for 3 minutes and separated bySDS/PAGE on 7.5% or 12% precast gels (BioRad Laboratories). For theimmunoprecipitated ¹²⁵I-labelled proteins the gels were stained with0.2% Coomassie Brilliant Blue in 25% isopropyl alcohol and 10% aceticacid, destained in 10% methanol and 10% acetic acid, dried and analyzedby autoradiography. For western blotting, the proteins separated bySDS/PAGE were transferred to nitrocellulose membrane (0.2 μ) for 16hours at 100 volts in 10 mM Tris-HCl pH 8.3, 76.8 mM glycine, 20%methanol and 0.01% SDS. The nitrocellulose membrane was blocked for 1hour at 37° C. in 3% gelatin, Tris-HCl pH 7.5, 0.15 M NaCl and thenprobed with hybridoma supernatant solutions or purified antibody dilutedin AB buffer (1% bovine serum albumin, 50 mM sodium phosphate pH 6.5,0.5 M NaCl, 0.05% Tween 20) for 16 hours at 4° C. After washing withwash buffer (PBS, 0.05% Tween 20), the nitrocellulose strips wereincubated for 2 hours at room temperature with goat anti-rat IgGantibody coupled to peroxidase (Boehringer Mannheim Biochemicals)diluted in AB buffer. The nitrocellulose membrane was washed with washbuffer and the bound antibody visualized by incubation for 30 minutes atRT with 4-chloro-1-napthol 0.5 mg/ml in 0.15% H₂O₂, 0.5 M NaCl, 50 mMTris-HCl, pH 7.5). The reaction was stopped by extensive washing withdistilled water.

In order that our invention herein described may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andshould not be construed as limiting this invention in any way to thespecific embodiments recited therein.

EXAMPLE 1

Purification of CLMF

CLMF was produced by a subclone of NC-37 lymphoblastoid cells aftercostimulation with PMA and calcium ionophore A23187. Stored, frozensupernatant solutions from these cells totaling 60 liters were thawed,pooled and concentrated to approximately 1.9 liters using the PelliconCassette System. To clarify this concentrate, the preparation wascentrifuged and the precipitate discarded.

The supernatant solution was applied to a Nu-Gel P-SP column and proteinwas eluted with a salt gradient (FIG. 1). Peak T cell growth factor(TGF) activity was determined and the active fractions were pooled anddialyzed in order to reduce the salt concentration of the preparation by50-fold. This material, after centrifugation to remove particulates, wasapplied to a Blue-B-Agarose column. Protein was eluted with a saltgradient (FIG. 2). Peak TGF activity was determined and the activefractions were pooled and dialyzed in order to reduce the saltconcentration of the preparation by 100-fold. This material afterfiltration, was applied to a Mono Q column. Protein was eluted with asalt gradient (FIG. 3). Aliquots of fractions were assayed for TGFactivity and protein purity of individual fractions was assessed bySDS-PAGE under non-reducing conditions using a 12% slab gel. The gel wassilver stained to visualize protein (FIG. 4). Fractions 36 and 37 wereof greater than 95% purity and revealed a major band at 75,000 molecularweight. Fractions 38 through 41 containing TGF in activity, revealed the75 kDa protein in by SDS-PAGE with major contaminants at 55,000 and40,000 molecular weight.

Therefore, to eliminate these contaminating proteins, fraction 38 of theprevious Mono Q chromatography was diluted 1:1 vol/vol with 8M urea andpumped onto a Vydac diphenyl column using an enrichment technique. Thecolumn was then washed with 5 ml of 0.1% trifluoroacetic acid. Elutionof the proteins was accomplished with a gradient of 0-70% acetonitrileover 7 hrs in 0.1% trifluoroacetic acid (FIG. 5). Aliquots of fractionswere assayed for TGF activity. Protein purity of the fractionscontaining TGF activity was assessed by SDS-PAGE under non-reducingconditions using a 10% slab gel. The gel was silver stained to visualizeprotein (FIG. 6). Fractions 86 through 90 were of greater than 95%purity and revealed protein of 75,000 molecular weight. Fractions 87 and88 were pooled and aliquots were analyzed by SDS-PAGE under reducing (inthe presence of β-mercaptoethanol and non-reducing conditions (in theabsence of β-mercaptoethanol). Under the reducing conditions, the 75,000molecular weight CLMF was separated into two subunits of 40,000 and35,000 daltons (FIG. 7). Thus we may conclude that CLMF is a 75 kDaheterodimer composed of disulfide-bonded 40 kDa and 35 kDa subunits.

The overall purification of CLMF that was achieved is shown in Table 1.The protein contest of the Mono Q- and Vydac Biphenyl-purified materialwas calculated on the basis of amino acid analysis. A specific activityof 8.5×10⁷ units/mg and 5.2×10⁷ units/mg for Mono Q- and VydacBiphenyl-purified material respectively, was obtained. The fact that thediphenyl-purified protein has a slightly lower specific activity thanthe Mono Q-purified material may be due to inactivation or denaturationof some of the molecules of CLMF in the HPLC elution solvents (i.e.,acetonitrile in 0.1% trifluoroacetic acid).

Chemical Characterization

The ability to prepare homogeneous CLMF allowed for the determination ofthe amino acid composition and partial sequence analysis of shenaturally occurring CLMF protein. Between 10 and 20 picomoles ofMono-Q-purified CLMF was subjected to hydrolysis, and its amino acidcomposition was determined (Table 2). Proline, cysteine and tryptophanwere not determined (ND). Quantitation of histidine was not possible dueto a large artifact peak, associated with Tris, coeluting with His (*).

Between 5 and 30 picomoles of diphenyl-purified CLMF was subjected tohydrolysis with and without pre-treatment with performic acid. Completeamino acid composition was thus obtained (Table 3) with the exception oftryptophan.

TABLE 1 Pooled Total Pooled Total Specific Volume Activity Units ProteinProtein Activity Step (ml) (U/ml) (U) (mg/ml) (mg) (U/mg) Pooled Cell60,000 2.58 × 10³ 1.6 × 10⁸ ND ND ND Supernatants Ultrafiltered 1,9401.57 × 10⁵ 3.0 × 10⁸ 1.83 3550 8.5 × 10⁴ Concentrate NuGel P-SP 90 2.00× 10⁶ 1.8 × 10⁸ 0.70 63 2.8 × 10⁶ Blue-B-Agarose 45 3.11 × 10⁶ 1.4 × 10⁸0.24 11 1.3 × 10⁷ Mono Q 1 6.40 × 10⁶ 6.4 × 10⁶ 0.075 0.075 8.5 × 10⁷Fraction 37 Mono Q 5 6.90 × 10⁶ 3.45 × 10⁷  0.081 0.405 8.5 × 10⁷Fraction 38 − >42 Diphenyl 1.1 5.74 × 10⁵ 5.22 × 10⁵  0.008 0.010 5.2 ×10⁷ Fraction 87 + 88

TABLE 2 Amino Acid mol % Aspartic acid or asparagine 11.8 Threonine 7.8Serine 8.4 Glutamic acid or glutamine 14.9 Proline ND Glycine 6.2Alanine 7.6 Cysteine ND Valine 6.9 Methionine 2.0 Isoleucine 4.6 Leucine9.0 Tyrosine 3.7 Phenylalanine 4.0 Histidine * Lysine 9.3 Arginine 5.4Tryptophan ND

TABLE 3 Amino acid mol % Aspartic acid or asparagine 10.8 Threonine 7.2Serine 8.9 Glutamic acid or glutamine 13.1 Proline 3.8 Glycine 4.7Alanine 5.9 Cysteine 2.9 Valine 6.2 Methionine 1.9 Isoleucine 4.2Leucine 9.4 Tyrosine 3.6 Phenylalanine 3.7 Histidine 1.8 Lysine 7.7Arginine 4.4 Tryptophan ND

Amino-terminal sequence determination was attempted by automated Edmandegradation on 100 pmol of the Mono Q-purified CLMF. Data from the first22 cycles indicated two sequences present, as would be expected from theheterodimeric structure of CLMF These results may be summarized asfollows:

Cycle 1 2 3 4 5 6 7 8 Amino I/? W/? E/L L/P K/V K/A D/T V/P Acid Cycle 910 11 12 13 14 15 16 Amino Y/D V/P V/G E/M L/F D/P W/? Y/L Acid Cycle 1718 19 20 21 22 Amino P/H D/H A/S P/Q G/? E/? Acid

EXAMPLE 2

Determination of the Amino-terminal Sequence of the 40,000 DaltonSubunit of CLMF

Stored supernatant solutions from NC-37 cells totaling 39.1 liters werepooled and concentrated to approximately 2.4 liters using the PelliconCassette System and stored at −20° C. To clarify this concentrate afterthawing, the preparation was centrifuged and the precipitate discarded.

The supernatant solution was applied to a Nu-Gel P-SP column and:protein was eluted with a salt gradient (FIG. 8). Peak TGF activity wasdetermined and the active fractions were pooled and dialyzed in order toreduce the salt concentration of the preparation by 50-fold. Thismaterial, after centrifugation to remove particulates, was applied to aBlue-B-Agarose column. Protein was eluted with a salt gradient (FIG. 9).Peak TGF activity was determined and the active fractions were pooledand dialyzed in order to reduce the salt concentration of thepreparation by 100-fold. This material, after filtration, was applied toa Mono Q column. Protein was eluted with a salt gradient (FIG. 10).Aliquots of fractions were assayed for TGF activity.

Fractions 39 and 40 of the previous Mono Q chromatography were pooledand diluted 1:1 vol/vol with 8M urea and pumped onto a Vydac Biphenylcolumn using an enrichment technique. The column was then washed with 5ml of 0.1% trifluoroacetic acid. Elution of the proteins wasaccomplished with a gradient of 0-70% acetonitrile over 7 hrs in 0.1%trifluoroacetic acid (FIG. 11). Aliquots of fractions were assayed forTGF activity. Protein purity of the fractions containing TGF activitywas assessed by SDS-PAGE under reducing (in the presence ofβ-mercaptoethanol conditions (FIG. 12). Fractions 94 through 97contained the 40,000 dalton subunit >90% pure.

Chemical Characterization

The ability to prepare a highly enriched preparation of the 40,000dalton subunit of CLMF allowed for its partial sequence analysis.

Amino terminal sequence determination was attempted by automated Edmandegradation on 20 pmol of the diphenyl- purified 40,000 dalton subunit.The results may be summarized as follows:

Cycle 1 2 3 4 5 6 7 Amino I W E L K K D Acid Cycle 8 9 10 11 12 13 14Amino V Y V V E L D Acid Cycle 15 16 17 18 19 20 21 Amino W Y P D A P GAcid Cycle 22 23 Amino E M Acid

With regard to the sequence analysis of 75,000 dalton CLMF (example 1)and the sequence analysis of the 40,000 dalton subunit of CLMF (example2), one can deduce the amino terminal sequence of the 35,000 daltonsubunit of CLMF. The amino terminal sequences of the 35,000 daltonsubunit and the 40,000 dalton subunit can be summarized as follows:

35,000 dalton subunit:                      5              10     NH₂-?-?-Leu-Pro-Val-Ala-Thr(?)-Pro-Asp-Pro-Gly-                   15                    20       Met-Phe-Pro-?-Leu-His-His-Ser(?)-Gln- 40,000 dalton subunit:         1               5                      10     NH₂-Ile-Trp-Glu-Leu-Lys-Lys-Asp-Val-Tyr-Val-Val-Glu              15                  20           23       Leu-Asp-Trp-Tyr-Pro-Asp-Ala-Pro-Gly-Glu-Met- where ?represents anundetermined or “best-guessed” residue.

EXAMPLE 3

Determination of Internal Amino Acid Sequence Segments of the 40,000dalton Subunit of CLMF

CLMF was purified as previously described in Example 1. The 40,000dalton subunit was separated and purified from the 35,000 dalton subunitby the method described by Matsudaira [Journal of Biological Chemistry262, 10035-10038 (1987)]. Fifty micrograms of CLMF (in 500 μl of 20 mMTris, pH 7.5; 0.15 M NaCl) was diluted with 200 μl 2× concentrateLaemmli sample buffer [Nature 227, 680-685 (1970)]. The sample wasconcentrated to 400 μl and disulfide bonds broken by the addition of 18μl β-mercaptoethanol followed by exposure to 105° C. for 6 minutes.

The sample was loaded onto a minigel (1.0 mm thick) containing 12%polyacrylamide and electrophoresed according to Laemmli. Afterelectrophoresis, the gels were soaked in transfer buffer (10 mM3-[cyclohexylamino]-1-propanesulfonic acid, 10% methanol, pH 11.0) for 5min to reduce the amount of Tris and glycine. During this time, apolyvinylidene difluoride (PVDF) membrane (Immobilon; Millipore;Bedford, Mass.) was rinsed with 100% methanol and stored in transferbuffer. The gel, backed with two sheets of PVDF membrane and severalsheets of blotting paper, was assembled into a blotting apparatus andelectroeluted for 30 min at 0.5 Amps in transfer buffer. The PVDFmembrane was washed in deionized H₂O for 5 min. The edge of the blot wasexcised from the PVDF membrane and stained with 0.1%. Coomassie BlueR-250 in 50% methanol for 5 min and then destained in 50% methanol, 10%acetic acid for 5-10 min at room temperature. The 40,000 dalton stainedband was then matched to the corresponding region of the unstained blotand the 40,000 subunit was cut from the unstained PVDF.

The Coomassie Blue-stained 40,000 dalton subunit was N-terminalsequenced to confirm that the N-terminus matched that previouslyobtained (see Example 2). By this method, the 40,000 dalton protein wasidentified as the 40,000 subunit of CLMF.

Five percent of the PVDF bound 40,000 dalton subunit was analyzed forits amino acid composition (Table 4). The remaining 95% of the blotted40,000 dalton subunit was fragmented with trypsin according to theprocedure of Bauw, et al. [Proc. Natl. Acad. Sci. USA 86,7701-7705(1989)]. The membrane carrying the protein was cut into pieces ofapproximately 3 by 3 mm, and collected in an Eppendorf tube. They werethen immersed in 300 μl of a 2% polyvinylpyrrolidone (40,000 dalton)solution in methanol. After 30 min., the quenching mixture was dilutedwith an equal volume of distilled water and further incubated for 5-10min. The supernatant solution was then discarded and the membrane pieceswere washed four times with 300 μl water and once with 300 μl 100 mMTris HCl (pH 8.5). Two hundred microliters of this buffer containing 2μg of trypsin was added. The sample was shaken and incubated for 4 hr at37° C. The supernatant solution was then transferred into a secondEppendorf tube and the membrane pieces were further washed once with 100μl of 88% (vol/vol) formic acid and three times with 100 μl of deionizedwater. All washing solutions were added to the digestion mixture in thesecond Eppendorf tube. The resultant peptides contained in the pooleddigest were separated by narrow bore HPLC (HP1090A, Hewlett Packard) ona YMC C-18 column (2.6×50 mm; Morris Plains, N.J.).

The above described procedure is shown, in cartoon form, in FIGS. 13 &14.

The tryptic peptide map of the digested 40,000 dalton subunit is shownin FIG. 15. Peptides were eluted with a linear gradient of acetonitrile.The peaks which were sequenced are numbered according to their fractionnumber. The amino acid sequence of these peptides is shown in Table 5.

Many tryptic peptides were recovered from all regions of the intact40,000 dalton subunit (Table 5). The N-terminal hexapeptide (fractionno. 60) was recovered in high yield. The carboxy-terminal peptide(fraction no. 72) was recovered and is the full length of the predictedC-terminal peptide although the last two amino acids were not positivelyconfirmed by sequencing. This is probably due to the fact that Cys andSer residues are not detected well, especially when they occur at theend of a peptide. Four potential Asn-linked carbohydrate sites arepredicted from the cDNA sequence. Two peptides containing two of thesesites were sequenced. When peptide 196-208 (fraction no. 70) wassequenced, no peak was detected at residue 200 indicating that this Asn(predicted by the cDNA) is indeed glycosylated. Peptide 103-108(fraction no. 52) yielded Asn at residue 103. Therefore, this site isnot glycosylated.

An unknown peak seen in the PTH (sequence) analysis of fraction no. 55was detected at the position corresponding to residue no. 148. The siteis predicted to be a Cys residue which is normally not detected bysequence analysis unless it is modified.

The above PVDF transfer procedure was repeated on a second 50 mg aliquotof CLMF (see FIGS 13 & 14 for procedure outline). However, the blotted40,000 dalton subunit was fragmented with the proteolytic enzyme,Staphylococcus aureus V8 protease (Endoproteinase Glu-C, BoerhingerMannheim, Indianapolis, Ind.). Membrane pieces were digested for 6 hoursat 37° C. with 20 mg of V8. The peptides were extracted with 88%(vol/vol) formic acid and separated on a Phase Separations column (2×150mm, C8 S3, Queensferry, England, UK) (FIG. 16). Peptides were elutedwith a linear gradient of acetonitrile. The peaks which were sequencedare numbered according to their fraction number. The amino acid sequenceof these peptides is shown in Table 6.

Three major peaks of peptide (fraction nos. 47, 54 and 57) containingfour peptides were sequenced. All four peptides were from theamino-terminal region of the 40 kDa subunit indicating that theN-terminus of the protein is most susceptible to V8-digestion.

FIG. 17 summarizes the protein structural determination of the 40,000dalton subunit to CLMF.

TABLE 4 Amino Acid Residue No. Aspartic acid or asparagine 27.9 (28)Threonine 20.7 (23) Serine 24.6 (34) Glutamic acid or glutamine 44.6(35) Proline ND (14) Glycine 16.3 (15) Alanine 16.2 (14) Cysteine ND(10) Valine 20.9 (23) Methionine 2.5 (2) Isoleucine 10.3 (12) Leucine22.9 (22) Tyrosine 12.9 (12) Phenylalanine 9.9 (9) Histidine 5.2 (5)Lysine 24.5 (26) Arginine 12.5 (12) Tryptophan ND (10)

Note: The results represent the mean of two analyses. Proline, cysteine,and tryptophan were not determined (ND). Values in parentheses representthe theoretical amino acid composition of the 40,000 dalton subunitbased upon the primary structure of the protein deduced from sequenceanalysis of cloned 40,000 dalton subunit.

TABLE 5 Tryptic 40kDa CLMF peptides off PVDF frac- resi- tion due no.no. N-terminal sequence 52 103- N-K-T-F-L-R 108 55 139-G-S-S-D-P-Q-G-V-T-*-G-A-A-T-L-S-A-E-R 157 55 & 267-V-F-T-D-K-T-S-A-T-V-I-?R 47 279(?) 57  52-58 T-L-T-I-Q-V-K 57 218-N-L-Q-L-K-P-L-K-N-S-R 228 60   1-6 I-W-E-L-K-K 67 288-? A-Q-D-R-Y-Y-S-S-67  85- K-E-D-G-I-W-S-T-D-I-L-K-D-Q-K-E-P- 102(?) 70 196-L-K-Y-E-?-Y-T-S-S-F-F-I-(R?) 208 71  85- K-E-D-G-I-?-S-T-D-I-L-K  96(?)72 288- A-Q-D-R-Y-Y-S-S-S-W-E-?-A-S-V-P-?-? 306(?) 78  71-85(G?)-G-E-V-L-S-H-S-L-L-L-(L?)-H-K-K

TABLE 6 V8 (Glu-C) 40kDa peptides off PVDF fraction no. residue no.N-terminal sequence 47 1-3 I-W-E 54  4-12 L-K-K-D-V-Y-V-V-E 57 13-22L-D-W-Y-P-D-A-P-G-E 57 45-59 V-L-G-S-G-K-T-L-T-I-Q-V-K-(E?)

EXAMPLE 4

Direct Determination of the Amino-Terminal Sequence of the 35,000 DaltonSubunit of CLMF

SDS-PAGE analysis of the Mono Q fraction 39 from Example 1 underreducing (in the presence of β-mercaptoethanol) and non-reducing (in theabsence of β-mercaptoethanol) conditions (FIG. 18) demonstrated that the40,000 dalton molecular weight “contaminant” is “free” 40,000 daltonCLMF subunit (i.e. unassociated with the 35,000 dalton subunit). Theevidence which points to this deduction is that without reduction (lane1, FIG. 18) mainly 75,000 dalton CLMF is present with some 40,000 daltonprotein. After reduction (lane 2, FIG. 18), the 75,000 dalton CLMF isgone yielding the 35,000 dalton subunit and an enriched 40,000 daltonband.

Fraction 39 of the previous Mono Q chromatography was reduced in 5%β-mercaptoethanol in the presence of 4 M urea and heated for 5 mins at95° C. The sample was pumped onto a Vydac C-18 column using anenrichment technique and the column was then washed with 5 ml of 0.1%trifluoroacetic acid. Elution of the proteins was accomplished with agradient of 0-70% acetonitrile over 5 hrs in 0.1% trifluoroacetic acid(FIG. 19). Protein purity of the fractions which were fluorescaminepositive was assessed by SDS-PAGE under non-reducing conditions using a10% slab gel. The gel was silver stained to visualize protein (FIG. 20).Fractions 112 through 117 revealed a diffuse band at 35,000 molecularweight which was greater than 95% pure. The 40,000 dalton subunit andany other proteins present in fraction 39 remained bound to the C-18column. These proteins (including the 40,000 dalton subunit) werefinally eluted with a solution of 42% formic acid/40% 1-propanol.

Chemical Characterization

The ability to prepare homogeneous 35,000 subunit allowed for thedetermination of the amino acid composition and partial sequenceanalysis of the lower molecular weight subunit of the CLMF protein.Approximately 1 μg of 35 kDa subunit was subjected to hydrolysis, andits amino acid composition was determined (Table 7). Proline, cysteineand tryprophan were not determined (ND).

Amino-terminal sequence determination was attempted by automated Edmandegradation on 100 pmol of the C-18 purified 35 kD subunit. Data fromthe first 20 cycles confirmed the sequence obtained by deduction asdescribed in Example 2. Furthermore, the second amino acid was obtainedin addition to amino acids 21 thru 26. These results may be summarizedas follows:

Cycle 1 2 3 4 5 6 7 Amino ? N L P V A T Acid Cycle 8 9 10 11 12 13 14Amino P D P G M F P Acid Cycle 15 16 17 18 19 20 21 Amino ? L H H S Q NAcid Cycle 22 23 24 25 26 Amino L L R A V Acid

Therefore, the amino terminal sequence of the 35,000 dalton subunit canbe summarized as follows:

35,000 dalton subunit:

                   5                      10NH₂-7-Asn-Leu-Pro-Val-Ala-Thr-Pro-Asp-Pro-Gly-Met-         15                  20                   25  26   Phe-Pro-?-Leu-His-His-Ser-Gln-Asn-Leu-Leu-Arg-Ala-Val

where ? represents an undetermined residue.

TABLE 7 Amino Acid Mol % Aspartic acid or asparagine 10.9 Threonine 6.7Serine 8.3 Glutamic acid or glutamine 14.9 Proline ND Glycine 6.1Alanine 7.7 Cysteine ND Valine 6.3 Methionine 2.9 Isoleucine 4.5 Leucine10.9 Tyrosine 3.2 Phenylalanine 4.4 Histidine 2.3 Lysine 5.6 Arginine5.5 Tryptophan ND

EXAMPLE 5

Determination of the Sequence of a Tryptic Fragment of CLMF

Mono Q fractions 36 and 37 from Example 1 were pooled (approximately 100pmol/1.7 ml) and the volume (less 30 μl-see Example 6) reduced to 200 μlunder a stream of helium. One hundred microliters of 0.1 M ammoniumbicarbonate was added. Trypsin (Worthington Biochemical Corp., Freehold,N.J.) cleavage was performed at a substrate-to-enzyme ratio of 2:1 (w/w)at 37° C. for 20 hr. The resultant peptide fragments were reduced andcarboxymethylated. This was accomplished by addition of 160 μl of 0.1 MTris-HCl, pH 8.5/6 M guanidine-HCl. The volume was reduced to 200 μlunder a stream of helium, and 4 μl of dithiothreitol (50 mg/ml) wasadded. The mixture was incubated at 37° C. for 4 hrs. After reductivecleavage of the disulfide bonds, [¹⁴C]iodoacetic acid (4 mmol) was addedand the resulting solution was incubated in the dark at room temperaturefor 10 min.

The resultant peptide fragments were isolated by reversed-phase HPLC(FIG. 21) on an S-5 120 Angstrom ODS column (2.6×50 mm, YMC, Inc.,Morris Plains, N.J.). Peptides were eluted with a 1-propanol gradient in0.9 M acetic acid, pH adjusted to 4.0 with pyridine. The amino acidsequence of the peptide found in fraction 46 was found to be (byautomated Edman degradation): Asp-Ile-Ile-Lys-Pro-Asp-Pro-Pro-Lys.

EXAMPLE 6

Determination of Internal Amino Acid Sequence Segments of CLMF

CLMF was purified as previously described in Example 1. Approximately 80μg of protein was precipitated with 10% trichloroacetic acid. Theprecipitate was dissolved in 70% (v/v) aqueous formic acid at roomtemperature. An approximately 50-fold molar excess over methionineresidues of cyanogen bromide (CNBr) in a small volume of 70% formic acidwas added, with stirring, and the mixture was incubated in the darkunder oxygen-free halium at room temperature for 48 hr. The mixture wasdiluted with 15 vol of water, divided into two equal portions and driedunder a stream of helium. For complete removal of the acid and byproducts, the drying was repeated after further addition of water.

One of the portions (@ 40 μg) of fragmented CLMF was dissolved with 50μl Laemmli sample buffer [Nature 227, 680-685 (1970)] containing 4%β-mercaptoethanol followed by exposure to 105° C. for 6 minutes. Thesample was loaded into 3 wells of a minigel (1.0 mm thick) containing17.5% polyacrylamide and electrophoresed according to Laemmli.

After electrophoresis, the gels were soaked in transfer buffer (10 mM3-[cyclohexylamino]-1-propanesulfonic acid, 10% methanol, pH 11.0) for30 min. During this time, a polyvinylidene difluoride (PVDF) membrane(Immolbilon; Millipore; Bedfor, Mass.) was rinsed with 100% methanol andstored in transfer buffer. The gel, backed with two sheets of PVDFmembrane and sandwiched with blotting paper, was assembled into ablotting apparatus and electroeluted for 30 min. at 0.5 Amps in transferbuffer. The PVDF membrane was washed in deionized H₂O for 5 min andstained with 0.1% Coomassie Blue R-250 in 50% methanol for 5 min, andthen destained in 50% methanol, 10% acetic acid for 5-10 min at roomtemperature. A number of smeared bands were observed.

Five regions of the membrane were excised across the three last lanescontaining the CLMF CNBr digest. These regions were sequenced. A summaryof the sequences obtained from the CNBr fragments of CLMF is shown onFIG. 22.

The second portion (@ 40 μg) of fragment CLMF was dissolved in −40-500μl 88% formic acid containing 6 M guanidine HCl, 0.1 M Tris/HCl, 0.5 MNaOH, pH 8.0. The sample was pH adjusted to pH 4.0 with formic acid. Thepeptide fragments were isolated by reversed-phase HPLC (FIG. 23) on aVydac C₄ column (4.6×20 mm, The Sep/a/ra/tions Group, Hesperia, Calif.).Peptides were eluded with a 4.5 hr linear gradient of acetonitrile in0.1% TFA. One of these peaks was sequenced and the amino acid sequenceof this peptide was:

Fraction No. N-Terminal Sequence 47 V-D-A-V-H-K-L-K-Y-E-?-Y-T-S-(S?)-F-F-I-R-D-I-I-K-P- (Starts at residue #190 of 40 #a subunit)

It is assumed or known that the above sequence is preceded by a Metresidue “?” represents a “best-guessed” residue.

EXAMPLE 7

Purification of CLMF and 40,000 Dalton Subunit Using AffinityChromatography

An affinity chromatography resin was prepared by covalently attaching7B2 monoclonal antibody to activated agarose. Similarly, the belowoutlined purification could also be carried out by covalently couplingthe antibody to silica or thin microporous membranes. The activatedagarose was prepared as follows:

1. 100 ml Sepharose CL-6B was washed three times with 100 ml H₂O.

2. 100 ml of 1% sodium meta-periodate in H₂O was added to the resin andthe suspension shaken at room temperature for 60 min.

3. The resin was washed with cold H₂O thoroughly.

The covalent attachment of 7B2 to the activated agarose was carried outas follows:

1. 9 ml of the activated agarose (described above) was suspended in 7 mlof 7B2 (@ 3.9 mg/ml) in phosphate buffered saline, pH 7.4.

2. Added 50.2 mg of cyanoborohydride was added to the gel suspensionwhich was shaken overnight at 4° C.

3. The gel suspension was filtered and added to 7 ml of 1.0Methanolamine, pH 7.0 containing 50.2 mg of cyanoborohydride.

One millimeter of the above described resin (@ 2.6 mg IgG/ml gel) waspacked in a column and washed extensively with phosphate bufferedsaline. Fractions from the Mono Q chromatography containing the 75 kDa,CLMF protein but with additional major contaminating proteins, werepooled (approx. 3.5×10⁶ U TGF activity) and dialyzed extensively againstPBS. This preparation was applied to the 7B2-Sepharose column at a rateof 5 ml/hr at room temperature. The column was washed with phosphatebuffered saline (pH 7.4) until baseline absorbance monitoring at 280 nmwas obtained. Adsorbed proteins were then eluded with 0.2 N acetic acid,0.15 M NaCl, pH @ 3. Aliquots of fractions were assayed for TGFactivity. Approximately 76% of the starting activity was recovered inthe acid eluate.

Protein purity was assessed without reduction by SDS-PAGE [Laemmli, U.K.(1970) Nature (London) 227: 680-685)] using a 10% slab gel. Gels weresilver stained [Morrissey, Anal. Biochem. 117:307-310] to visualizeprotein. The acid eluant contained pure CLMF and the “free” unassociated40 kDa subunit of CLMF (FIG. 24).

EXAMPLE 8

Determination of the pI of CLMF

Thirty microliters of the pooled Mono Q fractions 36 and 37 (see Example5) were spotted onto a precast ampholine PAGplate gel, pH 3.5-9.5(Pharmacia LKB Biotechnology) to determine the pI of CLMF. Based on pIstandard markers, a major band was observed at pI 4.8 and a minor bandat pI 5.2. Based on pH determination, the pI of these bands are 4.2 and4.6 respectively.

EXAMPLE 9

Biologic Activities of Purified CLMF

Purified CLMF stimulated the proliferation of human PHA-activatedlymphoblasts in the T cell growth factor assay (Table 8). The T cellgrowth factor activity of the purified CLMF recovered from the Mono Qcolumn was compared to that of a standard preparation of humanlymphokines in five separate experiments, and the specific activity ofthe purified CLMF was found to be 8.5±0.9×10⁷ units/mg protein. In oneexperiment in which purified CLMF obtained from diphenyl HPLC wascompared to the standard lymphokine preparation in the TGF assay, aspecific activity of 5.2×10⁷ units/mg protein was observed. Whensuboptimal concentrations of purified CLMF and human rIL-2 were testedin combination in the TGF assay, additive proliferation was observed(Table 8), up to the maximum proliferation caused by rIL-2 alone.However, proliferation caused by rIL-2 could be distinguished fromproliferation due to CLMF in that the former was totally inhibited inthe presence of a neutralizing goat anti-human IL-2 antiserum but thelatter was not affected.

The ability of purified CLMF to activate cytotoxic effector cells wasexamined both in a 4-day LAK cell induction assay and in an overnight NKcell activation assay. In the LCI assay, purified CLMF at concentrationsas high at 800 units/ml had little activity in the absence of IL-2(Table 9). However, CLMF synergized with low concentrations of humanrIL-2 in causing LAK cell induction in as much as the lytic activitygenerated in the presence of both cytokines was significantly greaterthan the sum of the lytic activities observed in cultures containingeither cytokine alone (Table 9). In the presence of rIL-02, purifiedCLMF was active at concentrations as low as 3 units/ml.

In contrast to the results in the 4-day LAK induction assay, purifiedCLMF was effective by itself in activating human NK cells in anovernight assay (Table 10). In this assay, CLMF was active atconcentrations as low as 1.6 units/ml. When CLMF was tested incombination with human rIL-2, the two cytokines together had, at best,additive effects in enhancing NK activity (Table 10).

In addition to its ability to enhance the lytic activity of nonspecificNK/LAK cells, CLMF also facilitated specific human cytolytic Tlymphocyte (CTL) responses in vitro. CLMF increased the specificallogeneic CTL response to weakly immunogenic, gamma-irradiated HT144melanoma cells (Table 11). In combination with a low concentration ofrIL-2, CLMF also facilitated specific allogeneic human CTL responses touv-irradiated HT144 melanoma cells, which did not elicit any detectableCTL response in the absence of added cytokines (Table 11). Thespecificity of the cytolytic effector cells generated in these studieswas demonstrated by their ability to cause substantial lysis of⁵¹Cr-labeled HT 144 melanoma cells but little or no lysis of K562 cells.In contrast, LAK cells which were generated in the same experiments byincubating low density lymphocytes with rIL-2 in the absence ofhydrocortisone lysed the K562 cells to a much greater extent than HT144melanoma cells. For further discussion of the specificity and identityof the cytolytic effector cells generated in assays such a those shownin Table 11, see Gately et al., J. Immunol. 136: 1274-1282, 1986.

Our results demonstrate that purified human CLMF by itself causedproliferation of activated human T lymphocytes, enhanced the cytolyticactivity of human NK cells, and augmented human CTL responses. Theseactivities of CLMF, which are similar to those of IL-2, suggest thatCLMF, like IL-2, should have immunoenhancing and antitumor effects whenused as a single therapeutic agent in vivo. Clearly, CLMF may also haveutility in stimulating the growth in vitro of NK/LAK cells and ofactivated T cells, such as may be derived from tumor infiltratinglymphocytes [S. L. Topalian et al., J. Immunol. 142: 3714-3725, 1989].In addition, purified CLMF synergized with low concentrations of rIL-2in causing the generation of human LAK cells in culture and actedadditively or synergistically with rIL-2 in facilitating specific CTLresponses in vitro. These results suggest that the use of CLMF incombination with rIL-2 might constitute a more optimal antitumortherapy.

TABLE 8 Purified Human CLMF Stimulates the Proliferation of HumanPHA-Activated Lymphoblasts Cytokine Added: ³H-Thymidine Incorporated byHuman CLMF^(c) Human rIL-2 PHA-Activated Lymphoblasts Expt. (u/ml)(u/ml) (mean cpm + 1 S.E.M.) 1^(a) 0 0 10,607 ± 596   500 0 70,058 ±1,630  100 0 60,377 ± 1,927  20 0 36,018 ± 321   4 0 24,996 ± 669   0.80 17,765 ± 790   2^(b) 0 0 9,976 ± 374   200 0 60,980 ± 1,713  50 038,817 ± 884   12.5 0 18,885 ± 2,132  3.1 0 13,648 ± 731   0 16 80,041 ±5,835  0 4 21,282 ± 1,145  0 1 11,241 ± 898   50 4 62,050 ± 2,408  12.54 40,628 ± 2,196  3.1 4 31,144 ± 3,754  ^(a)All cultures in experiment 1contained goat anti-human rIL-2. ^(b)No cultures in experiment 2contained goat anti-human rIL-2. ^(c)Purified human CLMF from Mono QFPLC.

TABLE 9 Purified Human CLMF Synergizes with Human rIL-2 in theGeneration of Lymphokine-Activated Killer (LAK) Cells in 4-Day CulturesCytokine Added: Human CLMF^(b) Human rIL-2 % Specific ⁵¹ Cr Release^(a)from: (u/ml) (u/ml) K562 Raji 0 0   3 ± 1.7   −1 ± 0.5   800 0   7 ± 0.3  1 ± 0.1 200 0   5 ± 1.1   1 ± 0.4 50 0   4 ± 3.0   0 ± 0.9 0 5  10 ±2.4   2 ± 0.8 800 5  41 ± 4.0  11 ± 0.8 200 5  42 ± 1.9  11 ± 0.3 50 5 36 ± 2.7   9 ± 0.8 12.5 5  28 ± 2.1   7 ± 0.7 3.1 5  19 ± 0.8   5 ± 0.30.8 5  14 ± 1.2   3 ± 0.8 ^(a)Values represent the means ± S.E.M. ofquadruplicate determinations. The spontaneous ⁵¹Cr release values forK562 and Raji were 16% and 14%, respectively. ^(b)Purified human CLMFfrom Mono Q FPLC.

TABLE 10 Purified Human CLMF Causes Activation of Natural Killer (NK)Cells in Overnight Cultures Cytokine Added: % Specific ⁵¹Cr Release^(a)from Human CLMF^(b) Human rIL-2 Raji Cells at Effector/Target Ratio =:(u/ml) (u/ml) 20/1 5/1 0 0 10 ± 0.6  5 ± 0.4 40 0 31 ± 0.4 14 ± 0.5 8 023 ± 2.1 12 ± 0.4 1.6 0 15 ± 0.3 10 ± 0.6 0.3 0 12 ± 1.2  9 ± 0.2 0 1 13± 0.4  6 ± 0.5 40 1 33 ± 2.0 17 ± 0.5 8 1 26 ± 0.8 13 ± 1.9 1.6 1 19 ±1.1 11 ± 2.1 0.3 1 16 ± 1.0 10 ± 1.5 0 5 20 ± 1.3 13 ± 0.6 40 5 23 ± 2.012 ± 1.5 8 5 29 ± 1.1 16 ± 0.7 1.6 5 27 ± 1.2 13 ± 0.8 0.3 5 24 ± 1.8 13± 1.2 0 25 38 ± 1.4 19 ± 0.7 ^(a)Each value represents the mean ± 1S.E.M. of quadruplicate determinations. The spontaneous ⁵¹Cr release was9%. ^(b)Purified human CLMF from Mono Q FPLC.

TABLE 11 PURIFIED HUMAN CLMF ENHANCES SPECIFIC HUMAN CYTOLYTIC TLYMPHOCYTE RESPONSES TO ALLOGENEIC MELANOMA CELLS IN VITRO Contents ofCultures: % Specific ⁵¹Cr Release from^(a) Lymphocytes HydrocortisoneMelanoma rIL-2 CLMF Expt. 1 Expt. 2 (Percoll fraction^(b)) (M) Cells^(c)(u/ml) (u/ml) HT144 K562 HT144 K562 4 10⁻⁴  6 ± 3 −4 ± 1 −2 ± 2 3 ± 4 410⁻⁴ 7.5 10 −3 ± 1 −1 ± 1 −5 ± 1 4 ± 1 4 10⁻⁴ HT_(uv)  0 ± 2 −3 ± 1 −2 ±2 2 ± 1 4 10⁻⁴ HT_(uv) 10  7 ± 2 −1 ± 1 13 ± 3 −4 ± 1  4 10⁻⁴ HT_(uv)7.5  5 ± 1  3 ± 1  1 ± 3 3 ± 3 4 10⁻⁴ HT_(uv) 7.5 10 20 ± 3 10 ± 1 27 ±9 −6 ± 1  4 10⁻⁵ HT_(γ) 17 ± 3 −4 ± 1 10 ± 1 3 ± 1 4 10⁻⁵ HR_(γ) 10 39 ±3 −2 ± 2 33 ± 6 5 ± 3 1 + 2 15 33 ± 4 67 ± 3 19 ± 1 47 ± 1  ^(a)In bothexperiments the contents of duplicate cultures were pooled, washed,resuspended in 1.2 ml TCM, and assayed for lytic activity undiluted andat 1:5 dilution. In experiment 1, the data shown were obtained using the1:5 dilution of lymphocytes in the cytolytic assay, corresponding to alymphocyte: target ratio of approximately 4:1. In experiment 2,significant lysis was seen only when lymphocytes were added undiluted tothe lytic assay, # and these data are shown in the table. Thespontaneous ⁵¹Cr release from HT144 cells was 25% and 31% in experiments1 and 2, respectively, and for K562 was 18% and 21% in experiments 1 and2, respectfully. ^(b)Percoll Fraction 4 contained high densitylymphocytes recovered from the interface between the 45% and 58% Percolllayers, whereas Percoll fraction 1 + 2 contained lower densitylymphocytes harvested from the interfaces between the 35% and 38% andbetween the 38% and 41% Percoll layers. Percoll fraction 4 contained CTLprecursors but few LAK precursors; on the other hand fraction 1 + 2 wasrich in LAK cell precursors. ^(c)HT_(uv) and HT_(γ) represent HT144melanoma cells which had been uv-irradiated or gamma-irradiated,respectively.

EXAMPLE 10

Cloning of a cDNA Coding for the 40 kDa Subunit of Human CLMF

1) Cell culture and isolation of polyA+ RNA

NC 37 cells (subclone 98) were grown in roller bottles as describedabove and induced with PMA and calcium ionophore for 15.5 hours. Thecells were harvested, resulting in a frozen cell pellet of 1.11 grams,5.25×10⁸ cells. A portion of the culture was continued for 3 days, atwhich the bioassay titer for CLMF activity read 2200 units/ml,indicating that the cells harvested for isolation of RNA had indeedproduced the CLMF activity. Total RNA was isolated from the frozen cellsby standard procedures and polyA+ RNA was obtained by affinitychromatography. The yield of polyA+ RNA was 2.5% (w/w) relative to thetotal amount of RNA input.

2) Establishment of a cDNA library

2 μg of the above polyA+ RNA were reverse transcribed into cDNA using150 ng random hexamers as primers. A library in lambda gt10 wasestablished, and 1.5×10⁵ clones were amplified for the screening.

3) Use of PCR to Generate a DNA Probe Specific for the 40 KDa CLMFSubunit cDNA

The partial N-terminal sequence of the purified 40 kDa protein isIWELKKDVYVVELDWYPDAP . . . . Two primers for use in mixed primer PCRwere designed and synthesized by standard procedures. The forward primerwas designed as the coding strand corresponding to amino acids ELKKD inthe above sequence, containing all possible codons for that sequence andhaving an extension at its 5′ end including an EcoRl site and threeadditional bases adding stability. The sequence of the forward primer isthus 5′ ctc gaa ttc gaa/g c/ttn aaa/g aaa/g ga, i.e. a 23mer with 64different sequences. The reverse primer was designed in the same manner,to represent the antisense strand corresponding to the amino acidsequence YPDAP in the partial N-terminal 40 kDa sequence. The reverseprimer thus has the sequence 5′ ctc gaa ttc ngg ngc a/gtc ngg a/gta andis a 24 mer containing 256 different sequences. The symbol n stands forany one of the four possible bases a,g,c or t. The primers thus definean amplicon of 72 basepairs in length. After cutting with EcoRl forgenerating cohesive ends for subcloning, the amplicon size drops to 64basepairs. Single-stranded cDNA was generated for use in the PCR asdescribed in section 2 above, using polyA+ RNA from induced and, as acontrol, uninduced cells. 40 ng of either one of those cDNAs wereamplified with forward and reverse primers in 100 μl of 10 mM Tris-HClpH 8.3/50 mM KCl/1.5 mM MgCl₂/0.01% gelatine/200 uM each of the fournucleotides/10 units Taq-polymerase/250 pmoles of each primer. The PCRparameters were as follows: initial denaturation was at 95° C. for 7minutes. Low stringency annealing was performed by cooling to 37° C.over 2 minutes, incubating 2 minutes at 37° C., heating to 72° C. over2.5 minutes, extending at 72° C. for 1.5 minutes, heating to 95° C. over1 minutes and denaturing at 95° C. for 1 minute, this low stringencyannealing cycle was repeated once. Afterwards, 30 standard cycles wererun as follows: 95° C. for 1 minute, 55° C. for 2 minutes. A finalextension was performed at 72° C. for 10 minutes. 10% of the totalsamples were run on a 4% agarose gel, stained and analyzed. The ampliconof the expected size was only detectable in the sample where inducedcDNA had been amplified. The remainder of the sample was extracted withphenol, concentrated by precipitation with ethanol and redissolved in 42μl of water. The sample was digested with 60 units of the restrictionenzyme EcoRl in 50 μl at 37° C. for 2 hours. The sample was subsequentlyrun on a 6% polyacrylamide gel and the 64 bp amplicon was cut out of thegel and eluted by standard procedures. The DNA amplicon was subclonedinto the EcoRl site of the bluescript SK+ plasmid by standard procedures(5). Colonies obtained from the transformation of the E.coli strain DH5alpha were picked and analyzed for the presence of the 64 bp insert. Twopositive candidates were sequenced to determine the sequence of thecloned amplicon. It is clear from this analysis that the correctfragment was amplified, since the deduced amino acid sequence matchesexactly the partial amino terminal amino acid sequence from the purified40 kDa protein. This information was subsequently used to design a 54 bplong oligonucleotide probe that could be used for screening of the cDNAlibrary. Two oligos were designed, with the following sequence: 5′ gagcta aag aaa gat gtt tat gtc gta gaa ttc gat and 5′ aag ggc atc cgg atacca atc caa ttc tac gac ata. These two oligos are partiallycomplementary to form the following structure:

5′ gagctaaagaaagatgtttatgtcgtagaattggat 3′

3′ atacagcatcttaacctaaccataggcctacgggga 5′

Such a structure can be labelled by using klenow fragment and labellednucleotides such that a high specific activity probe results for thescreening of cDNA libraries.

4) Screening of cDNA Libraries

A total of 3×10⁵ clones from the amplified library were screened on 6duplicate filters under the following conditions: 50 ml of 5×SSC/10×Denhardts/100 μg/ml denatured calf thymus DNA/20% formamide/0.1%SDS/1.5×10⁶ cpm of labelled 54 mer at 37° C. for 16 hours. The filterswere subsequently washed in 2×SSC at 42° C. for 30 minuted, dried andexposed to X-ray film. After overnight exposure with an intensifyingscreen, 16 possible positives were picked and further analyzed by asecond screening round, 10 rehybridizing phage were isolated and theirDNA prepared. 8 of those 10 ioslates looked identical, upon EcoRlcutting releasing two fragments of 0.8 kb and 0.6 kb length, indicatinga possible internal EcoRl site. Upon blotting and hybridization with thescreening probe, only the 0.6 kb fragment showed hybridization. The twofragments were subcloned separately into the EcoRl site of thebluescript SK+ plasmid as described above and were completely sequenced.This analysis showed that both fragments align in one contiguous cDNA ofabout 1.4 kb in length with a naturally occurring internal EcoRl site,since both fragments upon translation showed the presence of readingframes coding for tryptic peptides that had actually been isolated frompurified 40 Kd protein. The complete sequence of the 40 kDa subunit asdeduced from the cDNA is shown in FIGS. 25A-D. The cDNA codes for oneopen reading frame of 328 amino acids. The protein starts with theinitiating Met, followed by another 21 amino acids that make up aclassical hydrophobic signal peptide. The N-terminus of mature purified40 kDa subunit, i.e. IWELKKD . . . , follows immediately after thesignal sequence. The mature protein thus consists of 306 amino acids.The deduced protein sequences contains 4 possible N-linked glycosylationsites, two of which are present in isolated and sequenced trypticpeptides. One of these two sites is used in vivo for the attachment of acarbohydrate side chain. The molecular weight of the matureunglycosylated protein is 34699, the pI is 5.24. The corresponding mRNAis 2.4 kb in length and is detectable in a northern blot in steady stateRNA only from induced cells.

EXAMPLE 11

Cloning of a cDNA Coding for the 35 kDa Subunit of Human CLMF

Cell culture, isolation of mRNA and establishment of a cDNA library wereas described earlier for the cloning of the 40 kDa subunit.

Use of Mixed Primer PCR to Generate a DNA Probe Specific for the 35 kDaSubunit cDNA

The partial N-terminal sequence of the purified 35 kDa subunit is?NLPVATPDPGMFP?LHHSQNLLRAV . . . . Two primers for use in mixed primerPCR were generated by standard procedures. The forward primer wasdesigned as the coding strand corresponding to the amino acids DPGMF inthe above sequence, containing all possible codons for that sequence andhaving an extension at its 5′ end including an EcoRl site and threeadditional bases adding stability. The sequence of this forward primerwas thus 5′ CTC GAA TTC GAT/C CCN GGN ATG TT -3′, i.e. a 23 mer with 32different sequences. The reverse primer was designed in the same manner,to represent the antisense strand corresponding to the amino acids NLLRAin the partial N-terminal sequence. The reverse primer has the sequence5′ CTC GAA TTC NGC NCG/T NAA/G NAA/G A/GTT, i.e. a 24mer with 4096different sequences. In both primer sequences, N stands for all 4 bases.The two primers thus defined an amplicon 69 bases long. After cuttingwith EcoRl for generating cohesive ends for subcloning, the ampliconsize drops to 61 bases. About 3 μg of human genomic DNA were amplifiedwith forward and reverse primers in 50 μl of 10 mM Tris-HCl pH 8.3/50 mMKCl/1.5 mM MgCl₂/0.01% gelatine/200 μM each of the four nucleotides/2.5units of Taq polymerase/64 pmoles of forward and 2048 pmoles of reverseprimer (to compensate for the greatly differing complexities of the twoprimers). The PCR cycling parameters were as follows: initialdenaturation was at 95° C. for 7 minutes. Low stringency annealing wasperformed by cooling to 37° C. over 2 minutes, incubating at 37° C. for2 minutes, heating to 72° C. over 2.5 minutes, extending at 72° C. for1.5 minutes, heating to 95° C. over 1 minute and denaturing at 95° C.for 1 minute; this low stringency annealing cycle was repeated once.Afterwards, 40 standard cycles were run as follows: 95° C. for 1 minute,55° C. for 2 minutes and 72° C. for 3 minutes. A final extension wasperformed at 72° C. for 10 minutes. About 20% of the samples were run ona 6% polyacrylamide gel and an amplicon of the expected size wasdetected after staining the gel with ethidium bromide. The remainder ofthe sample was extracted with phenol, concentrated by precipitation withethanol and redissolved in 17 μl of water. The sample was digested with20 units of EcoRl enzyme in 20 μl for 60 minutes at 37° C. The samplewas subsequently fractionated on an 8% polyacrylamide gel and the 61basepair amplicon was cut out of the gel and eluted by standardprocedures. The DNA amplicon was subcloned into the EcoRl site of theBluescript plasmid SK+ by standard procedures. Colonies obtained fromthe transformation of the strain DH5 alpha were analyzed for thepresence of the 61 basepair insert. Two candidates were sequenced todetermine the sequence of the subcloned amplicon. One of the two clonescontained the correct sequence, since translation of that sequenceresulted in the amino acid sequence expected from the purified protein.Based on this information, two synthetic oligonucleotides were designed,with the following sequences:

5′gatccgggaatgttcccatgccttcaccactccc 3′

3′gtacggaagtggtgagggttttggaggatgcccga 5′

Such a structure can be labelled using radiolabelled nucleotides withthe Klenow fragment to very high specific activities for libraryscreening.

Screening of a cDNA Library:

A total of 10⁶ clones from the amplified 16 h library were screened on40 duplicate filters with the above probe under the followingconditions: 400 ml of 5× SSC/20% formamide/10× Denhardts/100 μg/mldenatured caly thymus DNA/0.1% SDS/3.8×10⁷ cpm labelled probe at 37° C.overnight. The filters were subsequently washed in 2× SSC at 40° C. andexposed to X-ray film with a screen overnight. Six potential positiveswere picked from this first round of screening and analyzed by a secondround of plaque hybridization, as above. One clone was picked for thefinal analysis. Upon preparing phage DNA, the clone was found to containtwo EcoRl fragments of about 0.8 kb and 0.3 kb in size. The twofragments were subcloned separately into the Bluescript SK+ plasmid andsequenced. This analysis showed that the two fragments align into onecontiguous sequence of about 1.1 kb total length with a naturallyoccurring internal EcoRl site. The complete sequence of the cDNA and thededuced amino acid sequence for the 35 kDa CLMF subunit are shown inFIGS. 26A-C. The cDNA codes for an open reading frame of 219 aminoacids, starting with the initiating Met at position 1. The following 21amino acids constitute a classical hydrophobic signal sequence.Immediately following the signal peptide, the N-terminus of the mature35 kDa protein starts with the sequence RNLPVAT . . . . Purified 35 kDaprotein had yeilded the sequence ?NLPVAT . . . . The mature 35 kDaprotein thus consists of 197 amino acids, containing three possibleN-linked glysocylation sites and 7 cys-residues. The molecular weight ofmature unglycosylated protein is 22513, the and pI is 6.09. Thecorresponding mRNA is 1.4 Kb in length and is only detectable in RNAfrom cells that had been induced for CLMF for at least 6 hours.

EXAMPLE 12

Expression of Biologically Active Recombinant CLMF in COS-cells

The two subunits for CLMF were engineered for expression in mammaliancells as follows:

40 kDa Subunit

The two EcoRl fragments constituting the full length cDNA for the 40 kDaCLMF subunit were ligated to an expression vector similar to pBC12 [SeeB. Cullen, Meth. Enzymology 152, 684,703, (1987)], except that the cDNAexpression is driven off the SV40 early promoter/enhancer. Clonescontaining the two inserts in the proper orientation to each other wereselected by colony hybridization with a synthetic oligonucleotide thatspans the internal EcoRl site in the 40 kDa cDNA. This oligonucleotidehas the following sequence: 5′ CTG AAG CCA TTA AAG AAT TCT CGG CAG GTG3′. It was labelled by kinasing using standard procedures. Clones weresubsequently analyzed for proper orientation of insert to vector by thepolymerase chain reaction procedure, using as forward primer a primerspecific for sequences in the SV 40 early promoter and as reverse primeran oligonucleotide corresponding to the 40 kDa cDNA sequence positionsno. 851-868. Clones with the correct orientation will give a PCRamplicon of 885 bp. Eight out of 20 clones tested gave the predictedfragment, and one was chosen for further study.

35 kDa Subunit

The full length cDNA for the 35 kDa subunit was amplified out of theoriginal lambda phage by PCR, using primers situated to the left andright of the EcoRl site in lambda gt10 (primers were New England BiolabArticles No. 1231 and 1232). The resulting PCR amplicon was blunt-endligated into the EcoRV site of the bluescript plasmid SK+ and the DNApropagated. DNA sequencing showed that the orientation of the cDNAinsert within the plasmid was such that the end of the cDNAcorresponding to the 5′ end of the mRNA was close to the ClaI site inthe polylinker. The insert was thus released by cutting with ClaI,filling out this end with T4 DNA polymerase and cutting secondarily withNot I. The resulting fragment was gel-purified and subcloned into anexpression plasmid based on the bluescript vector and containing theSV40 early promoter to drive expression of inserted cDNAs. The sites inthe expression plasmid used were a blunt-ended PstI site at the 5′ endand a Not I site at the 3′ end of the cDNA. One clone was chosen forfurther study after ascertaining its structure by PCR as above for the40 kDa construct.

Expression of the Two cDNAs in COS-cells

The DNAs for the expression constructs of the 40 kDa and 35 kDa subunitswere introduced into COS cells on 6 cm diameter plates by the DEAEDextran transfection procedure (7×10⁵ cells/dish plates; 1 μg DNA perdish). 24 hours after the transfection, the cells were fed with standardtissue-culture medium containing 1% Nutridoma instead of the fetalbovine serum and the supernatants were collected after 40 hours andfiltered through a 0.45μ filter.

Supernatant fluids from cultures of COS cells which had been transfectedwith cDNA encoding the 35 kDa CLMF subunit or the 40 kDa CLMF subunit orwith both cDNAs were tested for CLMF activity in the T cell growthfactor assay (Table 12). As shown in the table, COS cells which had beentransfected with only one of the subunit cDNAs did not releasebiologically active CLMF into the culture fluid. However, COS cellswhich had been transfected with both subunit cDNAs produced biologicallyactive CLMF. By comparing the amount of lymphoblast proliferationinduced by the culture fluid from doubly transfected COS cells to theamount of proliferation induced by purified NC-37-derived CLMF, theconcentration of CLMF activity, in the culture fluid was estimated to be374 units/ml. Assuming a specific activity of 8×10⁷ units/mg CLMFprotein, this result suggests that the fluid from cultures of doublytransfected COS cells contained approximately 4.7 ng/ml of recombinantCLMF.

TABLE 12 T Cell Growth Factor Activity of Recombinant CLMF Expressed inCOS cells ³H-Thymidine Incorporated by PHA-Activated LymphoblastsCytokine added: Concentration: (mean cpm ± −1 S.E.M.) None 14,587 ± 343Natural CLMF* 200 units/ml 79,848 ± 854 Natural CLMF 40 units/ml  59,093× 2029 Natural CLMF 8 units/ml 39,180 ± 545 Natural CLMF 1.6 units/ml25,996 ± 763 Supernatant fluid from cultures of COS cells transfectedwith: 35 kDa CLMF subunit cDNA 1/5 dilution 15,332 ± 797 35 kDa CLMFsubunit cDNA 1/25 dilution 12,149 ± 379 40 kDa CLMF subunit cDNA 1/5dilution  14,883 ± 1039 40 kDa CLMF subunit cDNA 1/25 dilution 13,889 ±110 35 kDa + 40 kDa CLMF subunit cDNAs 1/5 dilution 66,228 ± 166 35kDa + 40 kDa CLMF subunit cDNAs 1/25 dilution 47,873 ± 275 Mocktransfected 1/5 dilution 14,368 ± 628 Mock transfected 1/25 dilution14,426 ± 173 *Purified NC-37-derived CLMF from Mono Q FPLC.

EXAMPLE 13

Preparation, Characterization and Purification of Hybridoma Antibodies

Lewis rats (Charles River Laboratories, Wilmington, Mass.) wereinitially immunized by the intraperotineal route (i.p.) with partiallypurified CLMF mixed with an equal volume of Freund's complete adjuvant(Gibco). The rats were injected i.p. with booster immunization of CLMFmixed with Freund's incomplete adjuvant (Gibco) according to theschedule in Table 13. For preparation of activated spleen cells, one ratwas injected i.v. with partially purified CLMF on two successive daysstarting 4 days prior to the cell fusion (Table 14). Spleen cells wereisolated from this rat and fused with NSO cells at a ratio of 1:1(spleen cells: NSO cells) with 35% polyethylene glycol (PEG 4000, E.Merck). The fused cells were plated at a density of 5×10⁴ cells/well/mlin 48 well plates in IMDM supplemented with 15% FBS, glutamine (2 mM),betamercaptoethanol (0.1 mM), gentamycin (50 ug/ml), HEPES (10 mM) and15% P388D1 cell supernatant. Hybridoma supernatants were screened forspecific CLMF antibodies in 4 assays: 1) immunoprecipitation of¹²⁵I-labelled CLMF, 2) immunodepletion of CLMF bioactivity, 3) westernblotting with CLMF and 4) inhibition of ¹²⁵I-CLMF binding toPHA-activated PBL blast cells. Hybridoma cell lines secreting anti-CLMFantibodies were cloned by limiting dilution. Antibodies were purifiedfrom large scale hybridoma cultures or ascites fluids by affinitychromatography on protein G bound to cross-linked agarose according tothe manufacturer's protocol (Gammabind G, Genex, Gaithersburg, Md.).

Isolation and Identification of Monoclonal Antibodies Specific for CLMF

Serum isolated at the 3rd bleed from the rat immunized with partiallypurified CLMF (Table 13) neutralized CLMF bioactivity (5 units/ml) asdetermined in the TGF assay (FIG. 27). This neutralization could beblocked by adding excess CLMF (200 units/ml) demonstrating that theneutralization by the antiserum was specific for CLMF (FIG. 27). Normalrat serum did not neutralize CLMF bioactivity (FIG. 27). Spleen cellsisolated from this rat were fused with NSO Cells and the resultinghybridomas were initially screened for CLMF-specific antibodies byimmunoprecipitation of ¹²⁵I-labelled CLMF.

The radioiodinated partially purified CLMF preparation containspredominantly the CLMF 75 kDa heterodimer, a small amount of the freeCLMF 40 kDa subunit and two other proteins of approximately 92 kDa and25 kDa (FIG. 28). The ¹²⁵I-labelled CLMF preparation retained CLMFbioactivity in the TGF assay, indicating that the labelling proceduredid not significantly alter the configuration of the CLMF molecule. TheCLMF immunized rat serum immunoprecipitated the 75 kDa heterodimer andthe free 40 kDa subunit (Lanes 6 and 8, FIG. 28) whereas normal ratserum did not immunoprecipitate these radiolabelled proteins (Lanes 7and 9, FIG. 28). Four individual monoclonal antibodies alsoimmunoprecipitated the 75 kDa heterodimer and the free 40 kDa subunit(FIG. 28) but did not immunoprecipitate the 92 kDa or 25 kDa labelledproteins. The immunoprecipitation assay identified twenty hybridomaswhich secreted anti-CLMF antibodies (Table 14). All the antibodiesimmunoprecipitated the radiolabelled 75 kDa heterodimer and the free 40kDa subunit as determined by SDS/PAGE and autoradiography (data shownfor 4 representation antibodies in FIG. 28).

After initially identifying specific CLMF antibodies in theimmunoprecipitation assay, the antibodies were tested for their abilityto immunodeplete CLMF bioactivity as assessed by the TGF and LAK cellinduction assays. Increasing amounts of CLMF cause a dose dependentincrease in the proliferation of PBL blasts in the TGF assay as measuredby the incorporation of ³H-thymidine into the dividing blast cells (FIG.29). Immunodepletion of CLMF activity by immobilized anti-CLMFantibodies occurs in a dose dependent manner (FIG. 29). Aliquots (0.4and 0.1 ml) of hybridoma supernatant solution will completely deplete 50and 200 units/ml of CLMF activity from the culture medium. 0.025 ml ofsupernatant solution will completely deplete 50 units/ml but onlyapproximately 50% of 200 units/ml. 0.0062 ml of hybridoma supernatantshows even less depletion of 50 and 200 units/ml of CLMF. An aliquot(0.4 ml) of an anti-IL-1 receptor antibody supernatant solution shows noimmunodepletion of CLMF bioactivity.

Increasing amounts of CLMF also cause a dose dependent increase in thelysis of target cells by LAK cells as measured by the release of ⁵¹Cr inthe LAK cell induction microassay (FIG. 30). The immobilized anti-CLMFantibodies also deplete in a dose dependent manner the CLMF activity inthe LAK cell induction assay (FIG. 30). These data confirm that theantibodies which immunoprecipitate the 75 kDa labelled protein from theradiolabelled partially purified CLMF preparation are specific for CLMF.The data also demonstrate that the radiolabelled 75 kDa protein is theprotein responsible for CLMF bioactivity.

Identification of the CLMF Subunit Bound by the Monoclonal Antibodies

CLMF is a 75 kDa heterodimer protein composed of 40 kDa and 35 kDasubunits. Western blot analysis was used to determine if the monoclonalanti-CLMF antibodies recognized the 40 kDa or the 35 kDa subunits.Highly purified 75 kDa CLMF heterodimer was separated by non-reducingSDS/PAGE and transferred to nitrocellulose membrane (FIG. 31). Inaddition, purified CLMF, which was composed of approximately 95% free 40kDa subunit and 5% 75 kDa heterodimer, was separated by bothnon-reducing and reducing SDS/PAGE and the previous were transferred tonitrocellulose membrane (FIG. 32). Individual nitrocellulose stripscontaining the non-reduced 75 kDa CLMF heterodimer (FIG. 31), thenon-reduced 40 kDa subunit (top panel FIG. 32) and the reduced 40 kDasubunit (bottom panel FIG. 32) were probed with monoclonal anti-CLMFantibodies, control monoclonal antibody, rat anti-CLMF serum and controlrat serum. The monoclonal anti-CLMF and rat polyclonal anti-CLMFantibodies bind specifically to an approximately 75 kDa heterodimer onthe strips containing non-reduced 75 kDa CLMF while the control antibodypreparations do not show this binding activity (FIG. 31). All themonoclonal and rat polyclonal anti-CLMF antibodies recognize thenon-reduced 40 kDa subunit (top panel, FIG. 32). However, only the ratpolyclonal antiserum and three monoclonal antibodies, 8E3, 9F5 and 22E7,bind to reduced 40 kDa subunit protein (bottom panel, FIG. 32). Thesedata demonstrated that all the monoclonal antibodies were specific forthe 40 kDa subunit of CLMF.

Identification of a CLMF Receptor on PHA-Activated Lymphoblasts

The previous data demonstrated that the monoclonal anti-CLMF antibodiesimmunoprecipitated ¹²⁵I-labelled CLMF, immunodepleted CLMF bioactivityand bound to the 40 kDa subunit of CLMF. However, the antibodies presentin the hybridoma supernatant solutions could not be directly tested fortheir ability to neutralize CLMF bioactivity in the TGF or LAK cellinduction assays due to non-specific inhibitory effects of supernatantsolutions containing control antibodies. Our previous work with IL-2monoclonal antibodies demonstrated that antibodies which would block¹²⁵I-IL-2 binding to IL-2 receptor bearing cells would also neutralizeIL-2 bioactivity. Since receptor binding assays are usually unaffectedby addition of hybridoma supernatant solutions or other substances, aCLMF receptor binding assay was developed to evaluate the anti-CLMFantibodies for inhibitory/neutralization activity. A CLMF receptorbinding assay was configured with ¹²⁵I-labelled CLMF and thePHA-activated peripheral blood lymphoblasts (FIG. 33). The binding of¹²⁵I-CLMF to the PHA-activated lymphoblasts was saturable and specific(FIG. 33). Scatchard plot analysis [See Scatchard, G. Ann. N.Y. Acad.Sci. 51, 660-672 (1949)] of equilibrium binding data indicated that theapparent dissociation constant for ¹²⁵I-CLMF binding to the receptor isapproximately 200 pM and that each lymphoblast has about 700-800receptors. Since the serum from the rat immunized with CLMF showedneutralization of CLMF bioactivity, it was tested for inhibition of¹²⁵I-CLMF binding to the lymphoblasts (FIG. 34). The rat immune serumblocks 50% of ¹²⁵I-labelled CLMF binding at approximately a 1/500dilution, while the control rat serum does not show any inhibition atthis dilution. With the specificity of the receptor binding assayestablished, hybridoma supernatant solutions were tested for antibodieswhich would inhibit ¹²⁵I-CLMF binding to lymphoblasts.

The degree of inhibition of ¹²⁵I-CLMF binding to the lymphoblasts wasdetermined at a ½ dilution of each hybridoma supernatant solution (FIG.35). Twelve hybridoma supernatant solutions inhibited by greater than60% ¹²⁵I-CLMF binding to the lymphoblasts. The antibodies present inthese supernatant solutions have been classified asinhibitory/neutralizing antibodies. Six hybridoma supernatant solutionsinhibited ¹²⁵I-labelled CLMF binding by less than 40% and wereclassified as non-inhibitory/non-neutralizing antibodies. Controlantibody inhibited by approximately 10% the ¹²⁵I-CLMF binding to thelymphoblasts.

Three inhibitory antibodies, 7B2, 2A3 and 4A1, and two non-inhibitoryantibodies, 6A3 and 8E3, were purified from ascites fluid by protein Gaffinity chromatography on GammaBind G (Genex, Gaithersburg, Md.)columns. Antibodies 4A1, 2A3 and 7B2 inhibit in a dose dependent manner¹²⁵I-CLMF binding to the lymphoblasts with IC₅₀ concentrations of 0.7μg/ml, 7 μg/ml and 9.5 μg/ml, respectively (FIG. 36). Antibodies 6A3 and8E3 do not block ¹²⁵I-CLMF binding at concentrations of 100 μg/ml (FIG.36). These data demonstrated that the original classification of eachantibody as either inhibitory or non-inhibitory was correct.

Direct Neutralization of CLMF Bioactivity by Antibodies

To determine if the antibodies classified as inhibitory by the CLMFreceptor binding assay would directly neutralize CLMF bioactivity, eachinhibitory antibody was tested for neutralizing activity in the TGFassay (Table 15). Two inhibitory antibodies, 4A1 and 7B2, demonstrated adose dependent neutralization of CLMF bioactivity (40 units/ml) from0.03 to 100 μg/ml, with IC₅₀ concentrations of approximately 1 μg/ml and80 μg/ml, respectively. These data confirmed that antibodies inhibiting¹²⁵I-CLMF binding to the CLMF receptor would also neutralize CLMFbioactivity.

TABLE 13 Immunization Schedule: Total CLMF (10⁸ units/mg) Protein Spec.Activity Purity Date units mg (μg) (U/mg) (%)  3/28/89   1 × 10⁴  .1 μg15 6.7 × 10⁵ 6.7  4/10/89 1.2 × 10⁴  .1 μg ?   6 × 10⁵  .6  5/3/89 1stbleed  5/18/89 2.2 × 10⁵   2 μg 75 2.9 × 10⁶ 2.9  6/7/89 2nd bleed 6/29/89 6.3 × 10⁴ .63 μg 83 7.5 × 10⁵  .75  7/21/89 1.2 × 10⁵ 1.2 μg 24  5 × 10⁶ 5.0  8/2/89 3rd bleed 10/19/89 2.1 × 10⁶ (i.v.) 10/20/89 2.1 ×10⁶ (i.v.) 10/23/89 Fusion

TABLE 14 Monoclonal Anti-CLMF Antibodies (40 kDa Subunit Specific)Western Blot¹ ¹²⁵1-CLMF/Receptor Assay Neutralization Antibody Red. N.R.(% Inhibition)² of Bioactivity³ Inhibitory/Neutralizing 7B2 − ++ 95 +2A3 − ++ 99 + 1B8 − +/− 60 ND⁴ 1C1 − ++ 81 ND 4A1 − + 98 + 4C8 ND ND 68ND 4D1 − + 100 ND 6A2 − +/− 75 ND 7A1 +/− ++ 94 ND 8A1 − + 99 ND 8A2 −++ 83 ND 9C8 − + 62 ND 22E7 ++ ++ 91 ND Non-Inhibitory/Non-Neutralizing8E3 + ++ 35 − 9F5 + ++ 18 ND 4A6 − − 17 ND 6A3 − + 20 − 8C4 − ++ 33 ND8E4 − ++ 1 ND 39B3 ND ND 46 ND Control − − 12 − ¹Western blots: N.R. isnon-reduced and Red. is reduced SDS/PAGE For the western blots, a CLMFsample containing 5% 75 kDa heterodimer and 95% free 40 kDa subunit wereseparated on 10%. SDS/PAGE and western blots prepared as described inmethods. The blots were scored as strongly positive (++), positive (+),weakly positive (+/−), negative (−). ²CLMF receptor binding assay: Anantibody was considered inhibitory if it would block more than 60% ofradiolabelled CLMF binding to the PHA activated PBL blasts.³Neutralization of CLMF bioactivity as assessed by the TGF assay: Anantibody was considered neutralizing if it would block more more than50% proliferation at 200 μg/ml. The results are presented as positive(+) or negative (−). ⁴ND: Not Determined.

TABLE 15 Neutralization of CLMF Bioactivity by Monoclonal Anti-CLMF.Assay Contents: Total ³H-Thymidine CLMF^(a) Antibody^(b) Incorporation %Neutralization^(c) none none  9923 ± 439 CLMF none 25752 ± 592 CLMF 4A1100 μg/ml 12965 ± 938 81 20 12215 ± 663 86 4 12985 ± 269 81 .8 19932 ±1016 37 .16 22379 ± 410 21 .03 25405 ± 1093 2 CLMF 7B2 200 μg/ml 10763 ±878 96 100 15083 ± 406 67 20 23690 ± 1228 13 4 25849 ± 1408 0 CLMFControl 200 μg/ml 27654 ± 1086 0 100 22221 ± 381 22 20 27335 ± 620 0^(a)Purified CLMF was used in the TGF assay at a concentration of 40units/ml. ^(b)Purified antibodies were added at the concentrationsindicated in the table. ^(c)Reduction of ³H-thymidine incorporation tothe level seen in the absence added cytokines was considered to be 100%neutralization.

EXAMPLE 14

Preparation of Antibodies Against a Synthetic Peptide Fragment of the35,000 dalton Subunit of CLMF

A peptide, comprising amino acids 3-13 of the NH₂-terminal sequence ofthe 35 kDa CLMF subunit and a COOH-terminal cysteine(L-P-V-A-T-P-D-P-G-M-F-C), was synthesized by solid-phase peptidemethodology, purified by HPLC, and conjugated to keyhole limpethemocyanin via the methylated bovine serum albumin procedure. Two rabbiswere immunized intradermally with the conjugated peptide in Freund'scomplete adjuvant (300 μg peptide/rabbit). Six weeks after immunization,rabbits were boosted with free peptide (100 μg, intravenously) andKLH-conjugated peptide (150 μg, subcutaneously) dissolved in PBS. Serumsamples were prepared from bleedings taken 7 days later. The boostingand bleeding schedule was repeated every 4-5 weeks.

Serum samples from the first and second bleedings from each rabbit wereevaluated for reaction with the synthetic peptide in a direct ELISAassay. The synthetic, free peptide was coated on microtiter plates at 4ng/ml and 20 ng/ml, and the plates were washed and blocked with bovineserum albumin. Serum samples were tested at various dilutions (Table16), and antibody reactivity was detected with the use of a secondantibody (HRP-conjugated goat anti-rabbit IgG) with o-phenylenediamineas substrate. Absorbance values were read at 490 nm after addition ofH₂SO₄ to stop the reaction. The results indicate that antibody wasproduced in both rabbits against 35,000 dalton CLMF peptide (Table 16).In separate experiments, we verified that the antibody was specific forthe peptide since (a) serum from non-immunized rabbits does not reactwith the peptide in ELISA, (b) sera from rabbits immunized with thesynthetic peptide do not react with a peptide fragment from the 40,000dalton CLMF subunit and (c) purified IgG from the serum samples alsoreacts with the synthetic peptide.

A serum sample from one of the rabbits (first bleed) was tested byWestern blot analysis for reactivity with 75 kDa CLMF and with the 35kDa CLMF subunit (FIG. 37). Partially purified CLMF (approximately 120μg/ml) was run on SDS-PAGE, transferred to nitrocellulose, and treatedwith a 1:500 dilution of the rabbit anti-CLMF peptide antiserum.Antibody reactivity was detected by use of biotinylated goat anti-rabbitIgG and alkaline phosphatase-conjugated streptavidin. The anti-CLMFpeptide antibody was found to react both with nonreduced 75 kDa CLMFprotein and with the reduced 35 kDa CLMF subunit (FIG. 37).

Although the antibodies produced in this example were polyclonal, asimilar approach could be used to prepare monoclonal antibodies to the35 kDa subunit of CLMF. The synthetic peptide used in this example orother synthetic peptides based on the amino acid sequence of the 35 kDaCLMF subunit (FIG. 26) could be used to immunize rats. Fusions could beperformed and hybridoma cultures screened for the production ofmonoclonal anti-CLMF antibodies as described above.

TABLE 16 ELISA ASSAY OF SERUM SAMPLES FROM RABBITS IMMUNIZED AGAINST AKIT-CONJUGATED SYNTHETIC PEPTIDE FROM THE 35,000 DALTON SUBUNIT OF CLMFSerum Source A₄₉₀ Rabbit Bleed Free Peptide Serum Dilutions (fold) No.No. (ng/ml) 1 × 10⁴ 2.5 × 10⁴ 6.2 × 10⁴ 1.6 × 10⁵ 3.9 × 10⁵ 1 1 20 2.72.1 1.4 0.8 0.5 1 1 4 0.7 0.4 0.3 0.2 0.1 1 1 0 0.05 0.05 0.05 0.05 0.051 2 20 2.5 1.8 1.1 0.6 0.3 1 2 4 0.9 0.6 0.4 0.2 0.1 1 2 0 0.05 0.050.05 0.05 0.05 2 1 20 2.7 2.2 1.6 0.9 0.5 2 1 4 0.9 0.5 0.3 0.2 0.1 2 10 0.05 0.05 0.05 0.05 0.05 2 2 20 2.2 1.5 1.0 0.5 0.3 2 2 4 0.7 0.4 0.20.1 0.1 2 2 0 0.05 0.05 0.05 0.05 0.05

EXAMPLE 15*

Synthesis of IgE is dependent upon the balance between the production ofIL-4 and IFN-γ at the sites of T/B cell interactions (1-3). IL-4 maypromote IgE synthesis not only via a direct effect on B cells, bydirecting the switching to IgE (4-5), but also by regulating theproduction of other molecules or cytokines involved in IgE regulation.For example, IL-4-markedly inhibits IFN-γ production by humanlymphocytes stimulated by mitogen or allogeneic cells (6-7). Similarly,IFN-γ suppresses the in vivo synthesis of IgE not only by antagonizingthe effect of IL-4 on the switching to IgE but also by inhibiting theproliferation of IL-4-producing TH2 lymphocytes (9-10) or by directingthe differentiation of naive precursor T cells into T cells producingIFN-γ but not IL-4 (11). Lymphokines other than IL-4 and IFN-γ may alsohave an important role in the regulation of IgE synthesis. Interferon-α,a cytokine mainly produced by accessory cells, is a potent inhibitor ofthe in vitro and in vivo synthesis of mouse and human IgE (13). IFN-αalso counteracts the effect of IL-4 on the switching to IgE and mostinterestingly, like IFN-γ, it inhibits the in vivo production of IL-4and enhances that of IFN-γ (12). Interleukin-12 is a novel cytokine,which like IFN-γ and IFN-α, may be involved in protective immunityagainst infectious agents such as viruses. Also known as NKSF (NaturalKiller Cell Stimulatory Factor) or as CLMF (Cytotoxic LymphocyteMaturation Factor) IL-12 is a 75 kDa heterodimeric glycoproteindisplaying several in vitro activities including: (a) the enhancement,in synergy with IL-2, of the maturation of cytotoxic T cells and of LAKcells (lymphocyte activated killer cells); (b) the increase of thecytotoxic activity of NK cells (15); (c) the promotion of theproliferation of activated T cells and NK cells (16) and; (d) theinduction of IFN-γ production by resting or activated peripheral bloodNK cells and T cells (17). IL-12 is a strong inhibitor of the Tcell-dependent synthesis, of IgE by IL-4-stimulated peripheral bloodmononuclear cells and the IgE inhibition may be observed in the absenceof IFN-γ production.

*Provided by Dr. Guy Delespesse, University of Montreal.

Reagents

Human rIL-4 was obtained from CIBA-GEIGY, Basle, Switzerland (Dr. H.Hofstetter), and anti-CD40mAb 89 (18) was received from (ScheringPlough, Dardilly, France (Dr. J. Banchereau)); hydrocortisone wasobtained from Sigma (St. Louis, Mo.); PWM was from Gibco Laboratories(Grand Island, N.Y.); anti-IFN-γ neutralizing mAb was purchased fromGenzyme, (No. 1598-00 Boston, Mass.). In preliminary titrationexperiments, this antibody (25 μg/ml) completely neutralized thesuppressive activity of 500 IU/ml of IFN-γ on the IL-4-stimulatedsynthesis of IgE by PBMC. IgE (ng/ml) in IL-4-stimulated cultures was30±4 as compared to 9.8±2 in the presence of IFN-γ (500 IU/ml) and to31.7±3.8 in the presence of both IFN-γ and anti-IFN-γ mAb. Anti-LolplmAb is a mouse IgG1 antibody directed against the pollen antigen Lolpl(20).

Human rIL-12 and antibodies to IL-12

Human rIL-12 was produced by cotransfection of COS cells with a 1:1molar ratio of the two subunit cDNAs of IL-12 as described by Gubler etal. (13). Crude supernatant fluid from cultures of doubly transfectedcells was used as the source of rIL-12 in these experiments. Supernatantfluid from cultures of mock transfected COS cells was used as a control.Monoclonal anti-IL-12 antibody was a 1:1 mixture of two rat monoclonalanti-human IL-12 antibodies, 4A1 and 20C2, which were isolated andpurified as previously described (19). The 4A1 antibody is specific forthe 40 kDa subunit of human IL-12, and its isotype of IgG2b. The 20C2antibody appears to react with the 35 kDa subunit of IL-12, and itsisotype is IgG1. These two antibodies were previously found to synergizein blocking IL-12-stimulated proliferation of human PHA-activatedlymphoblasts.

Cell preparations and culture conditions

Cells were prepared and cultured as described (20,21): Briefly,peripheral blood mononuclear cells (PBMC) were isolated from heparinizedvenous blood of healthy individuals by centrifugation overFicoll-Metrizoate. Umbilical cord blood was collected inheparin-containing tubes and was sedimented 45 min at 37° C. withdextran (10% V/V; mol.wt. 200.000, Baker Chemicals Co., Phillipsburg,N.J.); the leukocyte-rich plasma was then layered on Ficoll-Metrizoate.Cells were cultured in HB101 culture medium (Hana Biologics Inc.,Alemeda, Calif.) supplemented with 5% fetal calf serum (Flow Labs,McLean, Va.) penicillin (100 U/ml), streptomycin (100 μg/ml, L glutamine(2 mM) (Gibco Laboratories), sodium pyruvate (10 mM) and Hepes (10 mM).Cells (2×10⁵ in 0.2 ml) were cultured in four replicates or more inround-bottomed 96-well tissue culture plates (Linbro) for 12 days in ahumidified atmosphere of 5% CO₂ and 95% air. For the induction of IgEsynthesis, cultures were supplemented with IL-4 at the finalconcentration of 10 ng/ml; this concentration was found to be optimalfor the induction of IgE synthesis and the suppression of IFN-γproduction in mixed lymphocyte cultures.

RIAs

Immunoglobulisn were measured in cell-free culture supernatants by meansof solid-phase RIAs exactly as described (20,21); IFN-γ was measured bya commercially available RIA (Centocor Co., Malvern, Pa.) with asensitivity of 1 U/ml. The net synthesis of Igs and of IFN-γ wasdetermined by subtracting the values measured in the culturesupernatants of cycloheximide-treated cells (50 μg/ml) from those ofuntreated cells. In preliminary experiments, where the levels of IFN-γin the supernatants of IL-12 stimulated cultures were determined at days2, 4, 6 and 8, we found, in agreement with a previous report (17), thata plateau was obtained between day 4 to day 6. IFN-γ was thereforeroutinely measured at day 6; to this end, 50 μl of culture supernatantswere collected and replaced by the same volume of fresh culture medium.All the culture supernatants were stored at −20° C. until the assay.

Northern blot analysis

Northern blot analysis was carried out exactly as described (6). TotalRNA was extracted from cultured PBMC by the guanidium-thiocynate methodwith CsCl gradient modification and quantified by measurement ofabsorbance at 260 nm. The samples (20 μg per lane) were subjected toelectrophoresis in formaldehyde-containing 1% agarose gel, andtransferred to nylon membrane (Biotrans, ICN, Irvine, Calif.). Themembrane was baked 2 h at 80° C. under vacuum, prehybridized in 50%formamide-5× Denhardt's-5× SSC-10 mM EDTA-50 mM sodium phosphate pH6.8-0.1%. SDS-250 μg/ml salmon sperm DNA and incubated overnight at 42°C. with ³²P labelled cDNA probe in the same buffer. The probes used forthe detection of the germ-line and the mature form of Cε mRNA weredescribed by Jaraba et al. (22). A 0.74 Kb Sma I fragment overlappingthe germline exon was used to detect germ-line Cε transcript, and the0.88 Kb Hinf I fragment encompassing most of the Cε1 exon and thetotality of the Cε2 exon was used to detect both the productive and thegerm-line Cε mRNAs.

IL-12 suppresses IgE synthesis

As seen in table 17, IL-12 (60 pM) significantly suppresses theproduction of IgE and increases the synthesis of IFN-γ by PBMC culturedin the presence of a saturating concentration of IL4 (10 ng/ml). IL-4significantly but incompletely suppresses the IL-12-induced productionof IFN-γ; and it totally abolishes the spontaneous production of IFN-γ.The effects of IL-12 on IgE and IFN-γ production are dose-dependent andthey are completely abolished by neutralizing anti-IL-12 mAbs (FIG. 38).The production of IgG, IgA and IgM in IL-4-stimulated cultures is notsignificantly affected by IL-12. However, IL-4 does not induce theproduction of IgM, IgA or IgG (with the exception of IgG4) (4). Theeffect of Il-12 on pokeweed mitogen (PWM)—induced IgE synthesis wasexamined. In 3 consecutive experiments, IL-12 (60 pM) had no significanteffect on the PWM-induced synthesis of IgG (1.6±0.1 versus 1.3±0.6μg/ml; mean±1 SD), IgM (1.2±0.4 versus 1.1±0.6 μg/ml) and of IgA(1.9±0.6 versus 2.1±0.7 μg/ml).

As shown in FIG. 39, IL-12 strongly suppresses the expression of themature but not the germ-line Cε transcript. This indicates that IL-12suppresses the synthesis of IgE and possibly inhibits the switching toIgE.

IL-12 appears to suppress IgE synthesis by a mechanism which is distinctfrom that of IFN-γ. The effect of IL-12 on the synthesis of IgE byumbilical cord blood mononuclear cells (CBMC) costimulated with IL-4 andhydrocortisone was tested. These cells were selected because of theirimpaired capacity to produce IFN-γ (23) and because exogenous IFN-γ wasfound to increase rather than to inhibit their synthesis of IgEfollowing stimulation with IL-4 (6). Hydrocortisone (HC) was added toIL-4 stimulated CBMC for two reasons: (i) HC inhibits the production ofIFN-γ, even that induced by IL-12; (ii) HC strongly increases the IL-4stimulated synthesis of IgE, even in the absence of IFN-γ production(24). As seen (table 18), IL-12 markedly inhibits IgE synthesis byneonatal cells cultured in the presence of IL-4 plus hydrocortisone andproducing little or nor detectable IFN-γ. Moreover, the suppression isunchanged in the presence of a large excess of neutralizing anti-IFN-γmAb. Thus, it appears that IL-12 can inhibit IgE synthesis by amechanism which is independent of IFN-γ.

As shown above, picomolar concentrations of IL-12 markedly inhibit thesynthesis of IgE by IL-4-stimulated PBMC. The suppression of IgE isobserved at the protein and the mRNA levels and it is completelyoverriden by neutralizing antibodies to IL-12. Given that the productionof IgE by IL-4-stimulated lymphocytes involves the switching ofprecursor B cells to IgE rather than the selective expansion anddifferentiation of IgE committed B cells (4, 25), the results suggestthat IL-12 inhibits the switching to IgE. consistent with anisotype-specific activity of IL-12, no influence is found of thislymphokine on the production of the other classes of Ig byIL-4-stimulated or by PWM-stimulated PBMC. The data do not exclude aneffect of IL-12 on the production of IgG4, the only human isotype otherthan IgE which is induced by IL-4 (4). Indeed, the measurement of IgG4,which is produced in very small quantities, requires the utilization ofIgG4 specific antibodies that are not employed in the RIA for IgGdetermination.

IL-12 induces the production of significant amounts of IFN-γ even in thepresence of a high concentration of IL-4, that was shown to completelysuppress IFN-γ production and to induce IgE synthesis in mixedlymphocyte cultures (6,7). Knowing that IFN-γ directs the in vitro aswell as the in vivo differentiation of naive T cells into TH1 type ofcells, it is reasonable to assume that IL-12 may display the sameactivity even in the presence of IL-4, which may also be produced bynon-T cells (26). According to this view, IL-12 might well pay a pivotalrole in determining the outcome of certain immune responses to certainantigens or pathogens that are known to preferentially generate TH1 orTH2 helper cells. The cellular origin of IL-12 is consistent with aputative role of this lymphokine in the differentiation of naive Tcells. Indeed, IL-12 may be produced not only by Epstein-Barr virustransformed B cells from which it was isolated but also by normal Bcells that are known to be efficient antigen-presenting cells.

In preliminary experiments using neutralizing antibodies to IFN-γ theIL-12 mediated suppression of IgE synthesis by adult PBMC was notconsistently overcome. These results may be easily explained by (i) therelatively high levels of IFN-γ in IL-12 containing cultures and (ii)the difficulty in blocking and biological activity of endogenouslyproduced IFN-γ. IL-12 also suppresses IgE by another mechanism which isIFN-γ independent. The existence of such a mechanism is demonstrated bythe ability of IL-12 to markedly inhibit IgE synthesis by IL-4 andhydrocortisone-costimulated neonatal lymphocytes which do not producedetectable amounts of IFN-γ. It is unlikely that such undectable levelsof IFN-γ (<1 IU) might nevertheless account for the suppression of IgEgiven that a very large excess of neutralizing anti-IFN-γ antibodyfailed to increase the IgE response. Whereas both IL-12 and IFN-γmarkedly suppress the accumulation of productive Cε mRNA inIL-4-stimulated PBMC (>90% suppression), IFN-γ, but not IL-12, alsosuppress the expression of germ-like trancript (50-70% inhibition). (5)IL-12 inhibits IgE synthesis of PBMC costimulated with IL-4 andanti-CD40 mAb, a model where IFN-γ was reported to be inactive (27). Inthree such experiments where PBMC were cultured with IL-4 and anti-CD40mAb 89 (0.5 μg/ml), the production of IgE dropped from 70±28 ng/ml(mean±1 SD) to 20±8 ng/ml in the presence of IL-12 (60 pM) as comparedto 79±35 ng/ml in the presence of IFN-γ (100 IU/ml). Taken collectively,the present results demonstrate that, like the interferons, IL-12 playsan important role not only in protective immunity but also in theregulation of isotype selection.

TABLE 17 EXP. 1 EXP. 2 ADDITION IgE IFN-γ IgE IFN-γ — <0.2 214 <0.2 62IL-4 57 <1 204 <1 IL-12 <0.2 3364 <0.2 2800 IL-4 + IL-12 19 1348 58 810

PBMC were cultured for 12 days in the absence or in the presence of IL-4(10 ng/ml), IL-12 (60 pM) or both. Shown are the mean values of IgE(ng/ml) and IFN-γ (IU/ml) measured in 4 replicate cultures; thevariation between the replicates was below 20%. Supernatant fluids fromcultures of mock transfected COS cells were inactive when used at thesame dilutions as the IL-12 containing supernatant fluids (not shown).

TABLE 18 EFFECT OF IL-12 ON IgE SYNTHESIS BY NEONATAL LYMPHOCYTESSTIMULATED WITH IL-4 AND HYDROCORTISONE EXP. 1 EXP. 2 EXP. 3 EXP. 4ADDITION IgE IFN-γ IgE IFN-γ IgE IFN-γ IgE IFN-γ — 373 <1 44 <1 20 <1302 <1 IL-12 6 <1 8 <1 <0.2 17 20 33 IL-12 + Anti- 7 <1 10 <1 <0.2 <1 21<1 IFN-γ IL-12 + Anti- 6 <1 NT NT NT NT 18 128 Lolpl Umbilical cordblood mononuclear cells were cultured for 12 days in the presence ofIL-4 and 10 μM hydrocortisone. IL-12 (60 pM), anti-IFN-γ mAb (1000neutralizing U/ml) or the isotype-matched control (anti-Lolpl) mAb (50μg/ml) were added at the initiation of the culture. IgE (ng/ml) andIFN-γ (IU/ml) were measured at day 12 and day 6 respectively. Shown arethe mean values of quadruplicate cultures; the variation between thereplicates was below 20%.

EXAMPLE 16

Provided below are several exemplary formulations for parenteral use,injection, or aerosol administration.

The following formulations are parenteral solutions. These solutions mayalso be lyophilized to form powders.

3.0 mg/ml CLMF is dissolved in phosphate-buffered saline pH 7 (PBS pH 7)q.s. 1 ml. To this solution may be added one of the following: 0.2 mg/mlPolysorbate 80; or 5.0 mg human serum albumin; or 10.0 mg/ml benzylalcohol. Alternatively, a formulation including 3.0 mg/ml CLMF; 25 mg/mlmannitol; and Tris buffer pH 7 q.s. 1 ml may be used.

A preferred formulation which is a lyophilized powder for injectionincludes 3.0 mg/ml CLMF, 3.0 mg/ml trehalose, and PBS pH 7 q.s. 1 ml.

All the above formulations are produced by mixing the indicatedcomponents in an appropriate vessel, filtering the resulting solution tosterility using an appropriate bacteria-retentive filter, andlyophilizing the resulting sterile solution in lyophilization vesselsusing an appropriate cycle. The lyophilization vessels should then bestoppered using the appropriate head pressure, gas, and stopper.

A suspension formulation for an inhalation aerosol may include thefollowing components: 1.5% w/w CLMF; 5.0% w/w mannitol; 2.0% w/wsorbitan trioleate; 64.0% w/w Freon 12; 11.5% w/w Freon 11; and 16.0%w/w Freon 114. This formulation may be produced by conventional means. Apreferred method of production is to prepare an aqueous solution of CLMFand mannitol and lyophilize this solution under suitable conditions. Tothe resulting dry product is added sorbitan triolate and Freon 11, andthe resulting mixture is homogenized. The resulting suspension is filledinto an aerosol container, which is crimped to seal with an appropriatevalve inserted. The container is then pressure-filled with an 80:20mixture of Freon 12 and Freon 114.

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What is claimed is:
 1. A pharmaceutical composition active in a T cell growth factor assay comprising a heterodimeric protein having a molecular weight of about 75 kD as determined by SDS-polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions, and under reducing conditions providing a first subunit having a molecular weight of about 40 kD as determined by SDS-PAGE, said first subunit having the following amino acid sequence Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu MET Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val MET Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro Cys Ser

and a second subunit having a molecular weight of about 35 kD as determined by SDS-PAGE, said second subunit having the following amino acid sequence Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly MET Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn MET Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe MET MET Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys MET Tyr Gln Val Glu Phe Lys Thr MET Asn Ala Lys Leu Leu MET Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn MET Leu Ala Val Ile Asp Glu Leu MET Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Thr Ser Tyr Leu Asn Ala Ser,

and a pharmaceutically acceptable carrier, said composition having a specific activity of from about 5.2×10⁷ units/mg to about 8.5×10⁷ Units/mg as determined in said T cell growth factor assay.
 2. The composition of claim 1, the protein being at least 95 percent pure heterodimeric protein as judged by silver staining following SDS-PAGE. 